β-Sitosterol was isolated from ethyl acetate leaf extract of Cissus populnea and characterized using H-NMR spectroscopy analysis. The antimicrobial activity of the leaf extract was also investigated in this study. Column chromatography was used to fractionate the ethyl extract, and thin layer chromatography was used to monitor the fractions. The result of the study showed that all isolates of Cissus populnea leaf inhibited or exhibited antibacterial and antifungal activities against tested microbes, with the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Cissus populnea fractions against tested microbes or organisms ranging from 6.25 to 100g/ml. The majority of the minimum inhibitory concentration (MIC) activity levels were lower than the MBC levels. A lower MIC value indicates that fewer plant fragments (isolates) are needed to prevent organism growth.; thus, plants with lower MIC scores are more effective antimicrobial agents.