Introduction: Fungal endophytes of medicinal plants origin are gaining increasing interest as sources of novel bioactive agents with therapeutic capacity. This study was designed to identify and evaluate secondary metabolites isolated from Fusarium equiseti; an endophytic fungus isolated from leaves of Ocimum gratissimum for their antimicrobial potentials. Methods: The isolation and molecular identification of the endophytic fungus, as well as fermentation, and extraction of secondary metabolites were carried out following standard laboratory procedures. The crude extract was partially purified by partitioning into different fractions using column chromatographic techniques and the fractions were tested for antimicrobial activity. The phytoconstituents in the bioactive fractions were detected by dereplication using High-Performance Liquid Chromatography linked with Diode Array Detection (HPLC-DAD). Results: Our findings showed that fraction 4 (DCM/ Methanol fraction 95:5) exhibited moderate to strong inhibition against the test micro-organisms namely, Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Candida albicans with MIC values in the range of 0.03 to 1 mg/mL but had no inhibition against Aspergillus niger and Salmonella typhi. The compounds detected in the HPLC assay include Phomoxanthone A, Scalarolide, equisetin, epi-equisetin an episomer of equisetin, Naamine A, Carbonarone A, destruxin B, cytosporin G, and WLIP (β-hydroxydecanoyl-Leu1-D Glu-De-Thr-Dval-DLeu3 DSer-Leu-Dser-Ile). These compounds have been reported to possess antimicrobial activity. Conclusion: The detection of these metabolites further confirms the potential of the endophytic fungus, Fusarium equiseti isolated from leaves of Ocimum gratissimum as a potential source of bioactive molecules for treatment of infectious diseases.