Harmful algal blooms pose a persistent threat to the integrity of freshwater ecosystems and public health. However, there are no selective chemical control agents available to suppress cyanobacterial growth without damaging beneficial phytoplankton. In this study, ten structurally diverse monoterpenes were assessed in vitro for their differential activity against the potent toxin-producing cyanobacterium Microcystis aeruginosa and the ecologically valuable microalga Chlorella sorokiniana using disc diffusion (DDM) and minimum inhibitory concentration (MIC) assays. Inhibition zones against M. aeruginosa ranged from 6.9 to 43.6 mm, with thymol recording the largest zone (43.6 mm). MIC values ranged from 0.25 to >1 mg/mL for both organisms, and selectivity indices identified camphor and carvone as the most cyanobacterium-preferential compounds, while carene and α-pinene showed the inverse selectivity pattern. Molecular docking against six AlphaFold2-predicted target proteins, photosynthetic complexes, Adenosine Triphosphate (ATP) synthase subunits, and superoxide dismutase (SOD) from both organisms, revealed binding affinities between −3.9 and −6.2 kcal/mol. Phenolic monoterpenes consistently engaged active-site glutamate and aspartate residues via hydrogen bonds and Pi–Anion interactions, most strikingly in the M. aeruginosa ATP synthase, whereas the M. aeruginosa SOD represented the least amenable target for all compounds. Computational ADMET profiling confirmed favorable pharmacokinetic properties and low predicted toxicity for the full panel.

Cyanobacteria are prolific producers of specialized metabolites of growing interest for blue biotechnology, transversal to various sectors such as cosmetics, foods and pharmaceuticals. In this work, the marine cyanobacterial strain Salileptolyngbya sp. LEGE 181209, from Cabo Verde, was systematically characterized to resolve its taxonomy, pigments profile, and cytotoxicity assessment. A polyphasic workflow combining 16S rRNA gene phylogenies, 16S–23S ITS secondary structures, p-distance, morphology, and scanning electron microscopy (SEM) was used to establish the taxonomic placement of the strain as a new species of the genus. PCR assays targeting the toxin biosynthetic genes mcyA and anaC, and cytotoxicity assays in HaCaT keratinocytes showed low-to-absent cytotoxicity, supporting a safety-forward profile for downstream use. A sequential extraction with solvents of different polarities yielded complementary pigment fractions profiled by HPLC-PDA and spectrophotometry. Total carotenoids reached 72.7 µg mg−1 of dry extract (DE), the profile being dominated by β-carotene and zeaxanthin (≈42 and 8 µg mg−1 of DE, respectively); chlorophyll-a was also very representative, reaching 85.6 µg mg−1 of DE. Phycobiliproteins dominated the polar fraction, with phycocyanin reaching 150 µg mg−1, followed by sugars (19.7 µg of glucose equivalents mg−1) and phenols (8.8 µg of gallic acid equivalents mg−1).

This study evaluates volatile extracts (HE1 and HE2) from the lichen Pseudevernia furfuracea as eco-friendly agents to control algal proliferation, specifically targeting the cyanobacterium Microcystis aeruginosa and the green microalga Chlorella sorokiniana. Both extracts exhibited potent anti-microalgal activity against the two species with a minimum inhibitory concentration (MIC) ranging from 375 to 750 µg/mL. Furthermore, both extracts reduced cell density by more than 98% after eight days of treatment. Chlorophyll a and protein levels decreased significantly (>80%) in both species, indicating suppression of pigment synthesis. However, their physiological responses were distinct: M. aeruginosa underwent early acute oxidative stress and severe membrane damage, while C. sorokiniana exhibited delayed oxidative activation and a negative growth rate, suggesting non-lytic metabolic inhibition. An in silico study by molecular docking of the most abundant compounds identified in these volatile extracts, such as terpenoids (abietatriene, δ-cadinene) and a phenolic compound (atraric acid), showed that these compounds interact with vital cellular targets in M. aeruginosa and C. sorokiniana and likely contribute to the effects observed in these two species. Predictive toxicity by applying the ADMET framework confirmed the favorable bioavailability and low acute toxicity of these volatile compounds. Therefore, P. furfuracea volatiles are promising, species-specific, and environmentally safe candidates for mitigating aquatic algal proliferation through targeted oxidative and metabolic interference.

Introdução: O impacto negativo, a nível ambiental e na saúde humana, dos corantes sintéticos é reconhecido por vários setores industriais, incluindo o têxtil, o alimentar e o cosmético, levando à procura de alternativas naturais mais seguras e sustentáveis1. As cianobactérias, consideradas os organismos mais antigos na Terra, são seres fotoautotróficos que podem estar presentes em diversos ambientes, mesmo em condições extremas. A produção de pigmentos por cianobactérias, como as clorofilas, os carotenoides e as ficobiliproteínas, tem sido amplamente estudada a nível mundial2. Objetivos: Este estudo teve como objetivo analisar as características dos pigmentos de cianobactérias para fins de coloração sustentável, destacando também os benefícios para a saúde decorrentes da sua aplicação. Metodologia: Realizou-se uma revisão da literatura, com recurso a várias bases de dados científicas, como a Web of Science, a b-on e o Google Scholar. A pesquisa foi realizada no ano de 2023, tendo sido incluídos os artigos enquadrados no tema, após análise do título e do resumo. Resultados e Conclusões: De acordo com os estudos analisados, os aspetos que mais contribuem para a vasta aplicação industrial dos pigmentos de cianobactérias estão relacionadas com: a sensibilização dos consumidores para opções mais sustentáveis, bem como a procura por produtos naturais; a disponibilidade de uma extensa paleta de cores; o risco reduzido de efeitos adversos para a saúde em comparação com os seus homólogos sintéticos; a conformidade com a regulamentação ambiental; e a ampla gama de propriedades bioativas. Em termos de benefícios para a saúde, no geral, os pigmentos de cianobactérias são descritos como compostos anti-inflamatórios, antioxidantes, anti-obesidade, antienvelhecimento, anti-cancerígenos, anti-microbianos, anti-diabéticos, neuroprotetores, hepatoprotetores e fotoprotectores, demonstrando a sua aplicabilidade na indústria farmacêutica, nutracêutica, alimentar e cosmética. Para além dos efeitos positivos na saúde humana, a utilização destes pigmentos tem um reduzido impacto ambiental quando comparado com os equivalentes sintéticos. Em suma, os pigmentos de cianobactérias revelam um elevado potencial como alternativas sustentáveis aos corantes sintéticos, conciliando segurança, funcionalidade e benefícios para a saúde com um menor impacto ambiental, o que reforça o seu valor estratégico para uma indústria mais verde e inovadora.

Microcystin (MC) contamination of surface waters threatens ecosystems and public health. Nature-based solutions such as Multi-Soil-Layering (MSL) systems have been used for MC remediation. However, the biological mechanisms controlling MC degradation remain unclear. The present study investigates microbial community responses in two MSL systems with different clay contents (8% and 54%) exposed to MC-contaminated inputs (well water and eutrophied lake water). Samples were analysed before and after treatment using quantitative PCR (qPCR) to quantify the mlrA gene (encoding microcystinase) and its bacterial hosts. Next-generation sequencing (NGS) was used to assess microbial diversity, while the FAPROTAX database was used to predict functional characteristics. Results showed that MC was mainly adsorbed in pozzolan layers, while mlrA gene abundance and MC-degrading bacteria were higher in soil mixture layers. The presence of mlrA and associated bacteria was most pronounced in lake inflow samples, indicating intrinsic MC Biodegradation potential. Taxonomic analysis revealed dominant phyla including Proteobacteria, Actinobacteriota, Firmicutes, Chloroflexi and Bacteroidota. Functional analysis identified dominant traits such as chemoheterotrophy and aerobic metabolism. These findings provide new insights into microbial interactions in MSL systems and contribute to the optimisation of water treatment strategies for MC-contaminated environments.

Nuclear receptors (NRs) of the NR1J1 group have been described in aquatic invertebrates and proposed as the evolutionary counterparts of vertebrate NR1I receptors, known for their role in xenobiotic-sensing and detoxification. In bivalves, previous studies have shown that NR1J1 activity and gene expression are modulated by pharmaceuticals, toxins, natural compounds, and algal extracts. However, their functional properties and diversification remain poorly understood. Here, we investigated the function and evolution of NR1J1 receptors in the bivalves Mytilus galloprovincialis and Ruditapes decussatus. We first identified four nr1j1 paralogs in R. decussatus and then integrated phylogenetic analysis, domain identity comparisons, tissue distribution profiling, and luciferase-based transactivation assays to characterize NR1J1 paralogs from both species. The receptors displayed distinct but partially overlapping transactivation profiles in response to confirmed ligands of NR1I and NR1H receptors, natural compounds, plant extracts, and fish bile, indicating paralog- and species-specific differences in ligand responsiveness. Allocholic acid emerged as the most consistent agonist across paralogs, whereas curcumin, carnosic acid, and Ptychopetalum olacoides extract showed more selective response patterns. Tissue profiling revealed broad but non-uniform expression of nr1j1 paralogs across bivalve tissues. Together, these findings underscore that bivalve NR1J1 receptors constitute a functionally diversified chemical-sensing system and provide a framework for future studies addressing ligand-dependent regulation and detoxification pathways.

Marine biofouling remains a major challenge for maritime industries, affecting submerged structures and vessels worldwide. The long-standing reliance on biocidal coatings, together with their documented environmental impacts, has led to increasingly restrictive regulations and an urgent demand for environmentally compatible antifouling (AF) solutions. This study evaluates the AF potential and toxicological profile of two nucleoside analogues, hypoxanthine arabinoside (1′) and 2′-deoxyinosine (2′), selected based on the previously reported non-lethal AF activity of the naturally occurring nucleosides adenosine and 2′-deoxyadenosine from cyanobacteria. Both analogues inhibited the growth of Navicula sp. by approximately 60% without inducing mortality and significantly reduced settlement of Mytilus galloprovincialis plantigrades, with EC50 values of 5.50 µM (1′) and 8.54 µM (2′), and no lethality detected (LC50 > 200 µM). At near-EC50 concentrations, both compounds increased acetylcholinesterase and tyrosinase activities, supported by molecular docking results, suggesting involvement of neurotransmission- and byssal formation-related pathways. Proteomic analysis revealed compound-specific molecular responses. No lethal effects were observed in non-target organisms (LC50 > 32 µM for A. amphitrite and LC50 > 50 µM for A. salina), and environmental fate modelling predicted low bioaccumulation and rapid degradation. Overall, substitution of the amino group by a carbonyl group preserved AF efficacy without increasing toxicity, highlighting nucleosides as promising low-toxicity AF agents.

The increase in multidrug resistance in microorganisms and the rise of emergent infectious diseases worldwide is a threat to human and animal health. Therefore, research on new molecules with antibiotic potential is a priority. Lichens have a unique secondary metabolism with relatively untapped potential, yet their essential oils (EOs) and volatile organic compounds (VOCs) remain a relatively untapped resource. This systematic review was conducted following PRISMA 2020 guidelines, with a comprehensive search performed in the Web of Science database for studies published up to 2023. From 254 identified records, six studies involving nine lichen species (Evernia prunastri, Evernia divaricata, Cladonia rangiformis, Cladonia furcata, Parmotrema perlatum, Lichina pygmaea, Parmelia perlata, Hypogymnia physodes, and Parmelia sulcata) met the eligibility criteria. The synthesized data show that these volatile fractions possess significant antimicrobial potential, with minimum inhibitory concentrations (MICs) generally lower than 1 mg/mL. Major bioactive constituents identified include atraric acid, orsellinates, and various sesquiterpenes. While the current evidence highlights a strong potential of lichen volatiles against pathogens, research is limited to a small fraction of known species. This review identifies a critical gap in testing these compounds directly against MDR clinical isolates and suggests that future research should focus on high-biomass species and the heterologous expression of lichen biosynthetic genes to develop sustainable antimicrobial applications.

Picocyanobacteria of the genus Synechococcus are a widespread component of marine planktonic communities and seasonally dominant in the Baltic Sea. Many Synechococcus strains have allelopathic properties, but the role of allelopathy remains unknown. Recent studies in 2-species systems provided experimental evidence for increased diversity at intermediate levels of allelopathy, while extreme levels (low or high) led to reduced diversity. Here, we studied the role of Synechococcus allelopathy in structuring coexisting phytoplankton communities. We brought natural phytoplankton communities from the Baltic Sea to equilibrium in semi-continuous cultures under nitrate limitation. These communities were inoculated with 2 Baltic Synechococcus strains (BA-124 and BA-132) known to have allelopathic properties. We used low, medium, and high cell densities as a proxy for allelopathy strength. With this design, according to classical resource competition theory, the dominant species at equilibrium should be the same irrespective of the initial inoculum of any species. Our equilibrium communities were dominated by 2-4 diatom genera at all Synechococcus levels. Community diversity was significantly higher only at the medium inoculum of strain BA-124, consistent with allelopathic effects shown in 2-species studies. There is no other clear factor that could produce such an effect, considering the experimental design. Therefore, we state that allelopathy is the best hypothesis to explain these results. In strain BA-132, the weaker allelopathic effect did not alter community diversity, with constant abundance in equilibrium indicating exploitative competition as the main driver of structure. These results suggest that allelopathy may be a driving factor of phytoplankton succession in the Baltic Sea.

The increasing demand for freshwater, in combination with climate change and pollution, compromises the quality and quantity of water resources. These pressures have intensified cyanobacterial blooms, including toxic events that threaten human health and aquatic ecosystems. This study complements the objectives of the Water Framework Directive by addressing the current lack of systematic data on toxic cyanobacteria and cyanotoxins. Therefore, the occurrence of toxic cyanobacteria in Mediterranean reservoirs was revisited, and the findings were complemented by case studies of two Portuguese reservoirs. Phytoplankton community composition and toxin concentrations were analysed, and cyanobacterial strains were screened for toxin-producing genes. The data gathered from literature revealed widespread cyanobacterial blooms across Spain, Portugal and Italy, with cell densities frequently exceeding the WHO medium-risk threshold (20,000 cells/mL) and occasionally reaching the very high-risk level (1000,000 cells/mL). Microcystins were the most frequently detected toxins, often occurring above the WHO guideline (1 mu g/L), whereas saxitoxin, anatoxin and cylindrospermopsin were detected less frequently. In Portugal, the Aguieira reservoir exhibited recurrent blooms and high phytoplankton biomass under eutrophic conditions, while the Rabag & atilde;o reservoir showed no relevant cyanobacterial proliferation. Molecular analyses confirmed the presence of the mcyA gene in Microcystis aeruginosa isolated from Aguieira, indicating its potential for microcystin production, whereas Aphanizomenon flos-aquae lacked detectable toxin biosynthesis genes. Overall, these findings highlight the ecological variability of cyanobacteria in Mediterranean reservoirs and emphasize the importance of integrating toxin monitoring and molecular tools into water quality assessments to strengthen management strategies and safeguard public health.

Biofouling—the adhesion of organisms and their byproducts to submerged surfaces—poses economic and environmental challenges, highlighting the need for sustainable antifouling solutions. This study reports a proof-of-concept investigation into the environmental compatibility and field validation of natural cyclic peptides portoamides A and B (Pam) as a bio-based antifouling alternative. Pam have demonstrated antifouling activity by inhibiting mussel larval settlement and disrupting biofilm formation. Herein, the antifouling performance of Pam-engineered coatings was evaluated through anti-settlement, anti-biofilm, as well as marine field tests. Lab-scale tests revealed that Pam-based coatings (0.7 wt%) effectively reduced biofilm thickness, surface coverage, and mussel larval settlement. Field trials showed that Pam-functionalized coating prototypes outperformed a commercial biocide in use (Econea®), delaying macrofouling community establishment and contributing to enhanced antifouling effectiveness. Overall, this work supports further development of antifouling engineered systems using Pam, representing a significant technological advance (from Technology Readiness Level 3 to 6) toward sustainable marine coating systems.

This article describes a new genus and species of cyanobacteria isolated from Laguna Pastos Grandes in the Bolivian Altiplano. This discovery marks the first described species of this phylum from this extreme environment. Additionally, the phylogenetic status of the genus Capilliphycus is reassessed. The analyses are based on 16S rRNA gene maximum likelihood and Bayesian phylogenies, identity (p-distance), morphology and habitat comparisons. The new genus is a Lyngbya-like type from a mat at the margin of a brackish–alkaline lake with borax. It is phylogenetically close to Dapis, Tenebriella and Okeania, but compared to these genera, the maximum 16S rRNA gene identity values reached only 90.9%, 91.1% and 92.5%, respectively. The genus Capilliphycus was found to be polyphyletic. The type species Capilliphycus salinus is clustered with C. guerandensis. However, “C. tropicalis” and “C. flaviceps” form a distinct clade, distant from the Capilliphycus type species. Between the two “Capilliphycus” clades, Sirenicapillaria and Limnoraphis are found.

White spot syndrome, caused by the white spot syndrome virus (WSSV), is a devastating viral disease responsible for significant economic losses in the shrimp farming industry. In the search for natural therapeutic alternatives against WSSV, this study explored the antiviral potential of portoamides, natural peptides produced by the cyanobacterium Phormidium sp. LEGE 05292. Given that the structural proteins of the viral envelope, specifically VP28, VP26, and VP24, are the primary mediators of host cell attachment, they serve as promising targets for antiviral drug development. Our approach combined in vivo post-infection histopathological analysis with in silico molecular docking to assess the antiviral efficacy of portoamides. In the in vivo study, crabs were injected with portoamides alongside WSSV and monitored for 30 days post-infection. The antiviral activity of portoamides was evaluated through survival rates and histopathological observations. The results revealed that crabs treated with portoamides showed improved survival and reduced signs of viral infection compared to the control group. In parallel, in silico molecular docking analysis was conducted to assess the binding affinity between portoamides and the viral envelope proteins VP28, VP26, and VP24. The docking results demonstrated that these proteins exhibited the highest binding energies with portoamides, indicating a strong interaction that could potentially inhibit viral attachment and replication. Our findings suggest that portoamides effectively inhibit WSSV replication by interacting with the viral envelope proteins, thereby preventing the virus from establishing infection in crabs. Moreover, it is hypothesized that portoamides may stimulate the immune system in crabs, further enhancing resistance to WSSV infection. However, additional studies are needed to fully understand the immunomodulatory mechanisms involved. These preliminary results highlight the potential of portoamides as natural antiviral agents for combating WSSV in aquaculture settings, paving the way for future research on their application in disease management strategies.

Cyanobacteria biodiversity remains underexplored despite their ecological importance and potential applications. To address this, we investigated two Leptolyngbya-like strains, LEGE 07170 and LEGE 191244, collected from marine tide pools in Portugal and Morocco using polyphasic approach. Phylogenetic analyses indicates that the strains form a distinct clade with strong statistical support in the Oculatellales order. The 16S rRNA gene identity matrix shows that the maximum shared values with the phylogenetically closest genera Gansulinema, Kaiparowitsia, Shahulinema, Aerofilum and Thermoleptolyngbya is consistently below 94.5%. Morphologically, LEGE 07170 and LEGE 191244 are indistinguishable to each other. Both optical and TEM analyses showed vacuole-like structures at the cells cross-walls and this character morphologically distinguishes these strains from their phylogenetically related genera. The 16S-23S ITS secondary structures also differed the LEGE-CC strains from their closest related genera.

Cyanobacteria are gram-negative prokaryotic microorganisms composed of both broad morphological and phylogenetic diversity inherited from diverse ecosystems like aquatic, terrestrial, or extremophilic environments. In this study, three cyanobacteria strains from the Blue Biotechnology and Ecotoxicology Culture Collection (LEGE-CC) were obtained from different environments in Portugal. Polyphasic analysis was applied for taxonomic identification. The proximate composition analysis indicated the lipid content (6.2% to 9.1% dry weight (DW)), protein content (28.2% to 62.9% DW), and carbohydrate content (19.5% to 46.1% DW). The fatty acid (FA) profiles of the strains revealed the presence of 19 different FAs, with FA 16:0 found in the highest abundance. The lipidomic analysis revealed 230 lipid species, with Laspinema sp. LEGE 06078 displaying the highest diversity (125 lipid species). These included species-specific and common lipids species that denote biochemical uniqueness that are also carriers of omega-3 FA (n-3). Biological assays exhibited strong antioxidant activity against ABTS•+ and DPPH• in Laspinema sp. LEGE 06078, while Sphaerospermopsis sp. LEGE 00249 was renowned for reducing lipids in zebrafish larvae. The findings are of immense significance on the lipidomics diversity of cyanobacteria in terms of nutrition, health, and biotechnology, such as addressing obesity and sustainable resource production.

A common assumption in the literature on information diffusion is that populations are homogeneous regarding individuals’ information acquisition and propagation process: Individuals update their informed and actively communicating state either through imitation (simple contagion) or peer influence (complex contagion). Here, we study the impact of the mixing and placement of individuals with different update processes on how information cascades in social networks. We consider Simple Spreaders, which take information from a random neighbor and communicate it, and Threshold-based Spreaders, which require a threshold number of active neighbors to change their state to active communication. Even though, in a population made exclusively of Simple Spreaders, information reaches all elements of any (connected) network, we show that, when Simple and Threshold-based Spreaders coexist and occupy random positions in a social network, the number of Simple Spreaders systematically amplifies the cascades only in degree heterogeneous networks (exponential and scale-free). In random and modular structures, this cascading effect originated by Simple Spreaders only exists above a critical mass of these individuals. In contrast, when Threshold-based Spreaders are assorted preferentially in the nodes with a higher degree, the cascading effect of Simple Spreaders vanishes, and the spread of information is drastically impaired. Overall, the study highlights the significance of the strategic placement of different roles in networked structures, with Simple Spreaders driving widespread cascades in heterogeneous networks and Threshold-based Spreaders playing a critical regulatory role in information spread with a tunable effect based on the threshold value. These effects have consequences to our understanding of social phenomena, such as the spread of innovations in heterogeneous social systems with the presence of eager (Simple Spreaders) versus averse (Threshold-based Spreaders) adopters, but also to information warfare on social media where Simple Spreaders can be seen as embedded agents (e.g., bots) used to amplify the virality of ill-intended content and, oppositely, Threshold-based Spreaders as an essential self-regulatory element of social systems operating as information filters.

Essential trace metals are vital for cellular processes, such as respiration, DNA replication, and photosynthesis. Cyanobacteria must tightly regulate metal homeostasis to prevent deficiency or toxicity, yet their metallophores remain overlooked. Here, we report lusichelins A-E (1-5), new metallophores isolated from the marine cyanobacterium Lusitaniella coriacea LEGE 07167. Their structures and configurational assignments were determined by using NMR, mass spectrometry, TD-DFT calculations, and retrobiosynthetic insights. Lusichelins feature a unique structural arrangement with thiazoline/thiazole rings connected via a vinyl group, an aliphatic carbon chain, or directly enabling the potential for metal coordination. Genomic analysis identified a hybrid PKS/NRPS biosynthetic gene cluster consistent with the lusichelin structure, bearing traits characteristic of metallophore biosynthesis. Functionally, lusichelins act as metallophores capable of chelating both iron and copper. Lusichelin C (3) consistently bound iron under both metal-rich and metal-limited culture conditions, while copper complexation was only observed under elevated copper levels. At physiologically relevant pH values, no significant metal-binding preference was detected. Moreover, compound production was maximized under metal-rich conditions and in response to copper limitation. Lusichelin B (2) exhibited cytotoxicity against colon carcinoma cells while reversing multidrug resistance via ABCB1 efflux pump modulation. These findings expand our understanding of cyanobacterial metallophores in microbial metal homeostasis and highlight their biological potential.

This study aims to reevaluate the diversity of 16S rRNA gene sequences associated with Scytonema (Nostocales, Cyanobacteria). For that, we first conducted a search for 'Scytonema' sequences available in GenBank (NCBI) and aligned the results with reference strains from other Nostocales genera. The alignment totalized 383 OTUs and 800 nucleotide positions analyzed. A FastTree phylogenetic analysis revealed that the 'Scytonema' sequences are distributed across 15 distinct clades in addition to the Scytonema sensu stricto clade. These additional clades are positioned among Nostocales genera and phylogenetically distant from the type species, S. hofmanni. Within these clades, we had access to the strain Scytonema sp. CCIBt3568, identified as S. schmidtii, which we believe should be separated from Scytonema, and described as a new genus in the future. Additionally, Bayesian Inference and Maximum Likelihood phylogenies were performed with 97 OTUs and 971 bp, and the polyphyletic status of Scytonema as currently recognized was confirmed.

Opuntia plants are well-known for their richness in numerous compounds with potential biological properties against human diseases, including ulcers, asthma, and hemorrhoids. This study aimed to evaluate the anticancer activity of different parts (seeds, pulp, and fruit) and extraction solvents of both cultivated and wild Opuntia fruits against various cell lines, as well as to identify the potential components responsible for this activity using a bioactivity-based molecular networking approach with LC-MS/MS data. Among the different Opuntia extracts, the aqueous wild seeds (WS) exhibited a potent cytotoxic effect (>35% of cytotoxicity) against the carcinogenic HCT-116, MG-63, and T-47D cells with minor toxicity (<15%) to immortalized non-cancerogenic normal cells (hCMEC/D3, HaCaT, and 3T3-L1). A notable sensibility of the colon carcinoma cancer line (HCT-116) was observed toward the aqueous WS, reducing their viability by 65.5% at the concentration of 1000 µg/mL, with an IC50 of 685.4 ± 24.3 µg/mL. This extract also displayed cytotoxic effects on HCT-116 multicellular spheroids, impacting only the outer layer of the spheroids containing the active proliferating cells. The metabolite profiling by LC-MS/MS on the GNPS platform identified 79 mass peaks; among them, 18 were specific to the aqueous WS, which could be related to the observed cytotoxic activity. Among them, some compounds were already reported to have anticancer properties, namely, americanin, xanthoxylol, syringaldehyde, vanillin, and sinapaldehyde. To conclude, Opuntia seeds may constitute an attractive source of several promising anticancer agents, paving the way for potential pharmacological exploitation to treat colon carcinoma.