Background Acute coronary syndrome (ACS) is a major cause of morbidity and mortality, and early diagnosis remains challenging. Stress- and inflammation-related biomarkers such as copeptin and neopterin may improve diagnostic and prognostic assessment. This study aimed to evaluate serum levels of copeptin and neopterin in patients with ACS compared with controls. Methods In this case-control investigation, a total of 130 patients diagnosed with ACS were included, comprising 54 individuals with ST-segment elevation myocardial infarction (STEMI), 26 with non-ST-segment elevation myocardial infarction (NSTEMI), and 50 with unstable angina pectoris (UAP). Additionally, 82 control subjects were recruited, consisting of 42 with stable angina pectoris (SAP) and 40 without any cardiac conditions (NCD). Blood samples were obtained alongside demographic information, and serum concentrations of neopterin and copeptin were quantified utilizing the ELISA technique. Results Serum copeptin concentrations were notably greater in the ACS patients (132.73 +/- 325.88 ng/mL) in comparison to the control group (87.22 +/- 218.36 ng/mL,p < 0.001). Similarly, serum neopterin levels were elevated in ACS patients (5.57 +/- 8.92 ng/mL) compared with controls (1.95 +/- 6.96 ng/mL, p < 0.001). Among the ACS subgroups, STEMI patients exhibited the highest copeptin (and neopterin) levels, whereas the NCD group had the lowest values (p = 0.001 for copeptin and p = 0.029 for neopterin). The multivariable logistic regression analysis indicated that smoking cigarettes (OR = 4.115, 95%CI: 1.759-9.625), levels of copeptin (OR = 1.013, 95%CI: 1.008-1.018), and neopterin concentrations (OR = 1.316, 95%CI: 1.112-1.558) were notably linked to a heightened risk of ACS. Conclusion Serum levels of neopterin and copeptin are markers of ACS presence and provide valuable information regarding disease severity and prognosis. These biomarkers may be effectively used alongside conventional markers to improve early diagnosis and guide therapeutic strategies in ACS patients.