Nature has been considered an interesting source of secondary bioactive compounds. Plants and their associated endophytes are common sources for these active constituents. Our study demonstrates the metabolomics profiling of the ethyl acetate extracts of three endophytic fungi associated with rosemary roots (Cladosporium spp., Alternaria spp. and Talaromyces spp.) in addition to the in vitro evaluation of the antitrypanosomal potential. The results revealed the presence of 47 metabolites from different chemical classes such as terpenes, phenolics, alkaloids, polyketides, macrolides, and others. Furthermore, the extracts of Cladosporium, Alternaria and Talaromyces exhibited potential inhibitory effects against T. brucei with IC₅₀ values of 1.3, 3.2 and 3.5 µg/mL, respectively. Supporting the study, the identified compounds were docked against two proteins: Rhodesain in complex with a macrolactam inhibitor and ornithine decarboxylase in complex with a c-terminal fragment of antizyme. The docking simulations showed that most of the identified compounds have moderate to comparable docking score (S = − 3.82 to − 6.10 kcal/mol) within rhodesain active site. In addition, they showed weak to moderate docking scores (− 2.33 to − 5.9 kcal/mol) with a differential docking profile within ornithine decarboxylase active site. According to these findings, fungal endophytes associated with rosemary roots can be considered as a promising source of antitrypanosomal bioactive metabolites.