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Endophytic fungi isolated from medicinal plants have an important role to play in the search for new bioactive natural compounds. However, despite their potential as repositories of bioactive compounds, the endophytes of African medicinal plants are largely underexplored. The aim of this study was to isolate and identify the endophytic fungi associated with Vitex payos and evaluate their antimicrobial and antioxidant potential. The surface sterilization technique was used to isolate the endophytic fungi that were identified by rDNA sequencing of the ITS region. Crude methanol and ethyl acetate extracts were screened for antimicrobial activity using the agar diffusion method and evaluated for antioxidant activity using a commercial total antioxidant capacity assay kit. The total phenolic content of the extracts was determined using the Folin–Ciocalteu method and functional groups present in the extracts were predicted using Fourier-transform infrared spectroscopy. Seven endophytic fungi isolates identified as Glomerella acutata, Epicoccum nigrum, Diaporthe species, Penicillium chloroleucon, Diaporthe endophytica, Mucor circinelloides, and Epicoccum nigrum were isolated from the tissues of Vitex payos. None of the extracts exhibited antimicrobial activity and the crude ethyl acetate extract obtained from E. nigrum demonstrated both the highest total phenolic content (2.97 ±0.13 mg GAE g−1 dry weight) and total antioxidant capacity (231.23 ±2.03 μM CRE). Fourier-transform infrared spectral analysis of the crude extracts from E. nigrum confirmed the presence of molecules carrying bonded hydroxyl functional group characteristic of phenolic compounds. These preliminary results indicate that most of the isolated fungal endophytes from V. payos belong to the phylum Ascomycota and that the isolated E. nigrum strain has potential as a source of natural antioxidants.