A novel endophytic fungal strain, Talaromyces verruculosus (AUMC15459), was successfully isolated from leaves of the medicinal plant Catharanthus roseus in Tripoli, Libya. The potential antimicrobial and antioxidant activity of the fungus was investigated. The ethyl acetate crude extract was assessed for its antibacterial activity against four standard pathogenic microbial strains: Bacillus subtilis (ATCC® 6633), Staphylococcus aureus (ATCC® 6538), Escherichia coli (ATCC® 25922),and Candida albicans (ATCC® 10231). Furthermore, the ethyl acetate crude extract showed an in vitro growth inhibition of all tested pathogenic bacteria and Candida species. Moreover, the antioxidant activity was assessed using a DPPH radical scavenging assay with a recorded IC₅₀ of 277.9 µg/mL. Additionally, GC-MS was conducted to identify the effective compounds in the fungal ethyl acetate crude extract. The most predominant compounds found in the ethyl acetate crude extract, Butyl-2-ethylhexyl phthalate and Heptadecene-(8)-carbonic acid-(1), are known for their antibacterial and antioxidant activities. The endophytic Talaromyces verruculosus (AUMC15459) was submitted to GenBank under the accession codeOR755888. The identified isolate is a prospective strain with efficient metabolites. To the best of our knowledge, this study represents the initial discovery of the broad-spectrum antimicrobial and antioxidant efficacy of the endophytic T. verruculosus AUMC15459 crude extract for potential applications in the medicinal field.