Powdered stem bark of plant material (1000g) of Diospyros mespiliformis was extracted with hexane using Soxhlet apparatus and concentrated on a water bath at 60 0 C. The extract was later subjected to column chromatography techniques using gradient elution in which various fractions were obtained and monitored using thin layer chromatography. The structure of the isolated compound were suggested by 1D and 2D spectroscopic analysis and by direct comparison of the data obtained with those reported in literature to be Lupeol.Antimicrobial screening of the lupeol compound and control drugs were carried out using agar well diffusion method. Significant zones of inhibition of lupeol compound were observed from (30-24mm) and standard control drugs range from (40-32mm). Minimum Inhibitory Concentration(MIC) of the lupeol compound was observed at 6.25-12.5ug/ml and Minimum Bacteriacidal Concentration (MBC) /Minimum Fungicidal Concentration (MFC) ranged from (12.5-25ug/ml).Lupeol compound revealed significant antimicrobial activity against some selected clinical isolates in this study and thus validated the traditional claims ofDiospyros mespiliformis used for the treatment of antimicrobial based infections.