Nelsonia canescens is traditionally used to treat infections. This study evaluated its phytochemical composition and antimicrobial activity to validate ethnomedicinal claims. The methanol extract and fractions (hexane, chloroform, ethyl acetate, and butanol) were screened for phytochemicals and tested against Staphylococcus aureus, Escherichia coli, Salmonella typhi, Candida albicans, and Aspergillus niger using agar diffusion and broth dilution methods, with ciprofloxacin and fluconazole as controls. Phytochemical analysis revealed flavonoids, saponins, tannins, alkaloids, and cardiac glycosides. Antimicrobial assays showed that the butanol fraction (BF) and an isolated compound (IS) exhibited strong activity, with inhibition zones of 30–36 mm (S. aureus) and 28–30 mm (E. coli). The ethyl acetate fraction (EAF) showed moderate antifungal effects (17 mm against C. albicans). IS had the lowest MIC values (2.5–10 mg/ml), comparable to ciprofloxacin (1.25–2.5 µg/ml), and demonstrated bactericidal effects (MBC: 5–50 mg/ml). Non-polar fractions (CME, HF, and CF) were inactive, while fluconazole was effective only against fungi (MIC/MFC: 1.25–2.5 µg/ml). Polar fractions (BF, EAF) and IS displayed promising antimicrobial activity, particularly against Gram-positive bacteria and C. albicans, though standard drugs were more potent. The presence of bioactive compounds supports N. canescens traditional use, highlighting its potential as a source of natural antimicrobial agents. Further optimization could enhance efficacy.