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Heterotis rountidifolia (Sm.) Jacq. Fel, is employed in Nigeria traditional medicine for the treatment of various diseases. The study investigated the antioxidant, mineral composition and antimicrobial activities of aerial part of Heterotis rotundifolia using standard procedures. The proximate composition on dry matter basis showed high carbohydrate (48.41%) and low lipid (0.77%) contents while mineral content revealed that sodium (5.68 mg/100 g) and zinc (4.64 mg/100 g) were the highest. The total caloric value per 100 g was 260.93 kcal. Methanol (ME) fraction exhibited the highest radical scavenging (EC50 = 59.36 µg/mL) and reducing (EC50 = 76.54 µg/mL) activity. Contents of total flavonoids and phenolics were highest in ME and ethyl acetate (EAE) fractions (39.2 mg GAE/g and 183.5 mg RE/g, respectively), while hexane (HE) fraction showed the lowest radical scavenging, ferric reducing and nitric oxide assay (EC50 = 268.56, 87.86, and 98.30 µg/mL, respectively). The minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) against various bacterial and fungal strains using tube dilution method showed strong activity at MIC of 50 mg/mL depicted by n-HE fraction, though lower MBC of 37.5 mg/mL by ME fraction made the ME fraction of better potentials. Gas chromatography-mass spectrum (GC-MS) analysis of the ME fraction revealed 5-Hydroxymethylfurfural (16.87%), Methyl 6-O-[1-methylpropyl]-β-D-galactopyranoside (16.07%), 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy- (8.28%), 6-methyl-β-D-glucopyranose, 1,6-anhydro- (6.56%), 2-heptanol (6.82%), stigmastan-3,5-diene (4.32%) amongst others. This research demonstrates that extract and fractions from aerial part of H. rotundifolia possesses antioxidant, antimicrobial and nutritive potentials, albeit with generally weak antifungal activity and, may be attributed to the presence of its phytochemical constituents. [ABSTRACT FROM AUTHOR]