Khaya senegalensis is a plant widely used in Benin’s traditional medicine for treating various diseases. Due to overexploitation, it faces anthropogenic pressure, threatening its extinction. This study aimed to evaluate the enzymatic and antimicrobial activities of endophytes in Khaya senegalensis. Fresh leaves, stems, and roots collected from Abomey-Calavi were sterilized and cultured on specific media to isolate bacterial and fungal endophytes. The isolation rate of these endophytes was determined. Their enzymatic activities (amylase, lecithinase, lipase, cellulase) were explored, and the antimicrobial activity of these endophytes against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans was evaluated by agar diffusion. The results revealed that the leaves of Khaya senegalensis had the highest endophyte isolation rate, with Bacillus spp. (46.67%) dominating among bacteria and Alternaria spp. (35.71%) predominating among fungi. All isolated bacterial endophytes showed varied enzymatic activities (catalase, amylase, lecithinase, hemolysin) but no antimicrobial activity against the tested strains. However, the fungal endophytes inhibited bacterial strains to varying degrees but had no effect on Candida albicans. Fungal isolates F1c, F3a, and T2c exhibited antibacterial activity against all tested bacterial strains, with F1c showing the greatest potential. This study highlights the diversity of bacterial and fungal endophytes in Khaya senegalensis, which exhibit different enzymatic and antimicrobial profiles.