Phylogenetic and functional genomic analyses were carried out to help explaining the endophytic lifestyle and the biocontrol activity of Streptomyces sp. strain DEF39, able to reduce Fusarium graminearum infection as well as deoxynivalenol production in wheat plants. Specifically, in this work, genes and biosynthetic pathways linked to secondary metabolite production, biocontrol activity, and plant interactions were identified. This approach supports the experimentally validated capability of DEF39 to protect plants from fungal diseases, highlighting the potential of genomics studies. Streptomyces sp. DEF39 was isolated as an endophyte from Secale cereale in Italy and has been shown to colonize wheat (Triticum spp.) and act as a biocontrol agent of Fusarium head blight. The complete genome (9.12 Mb) (GCA_978019405.1), assembled through a hybrid strategy based on long and short accurate reads combination, is composed of a linear chromosome of 8,887,323 bp with a GC content of 71.59