Abstract: The main objective of this study was to isolate compounds from root wood of Moringa stenopetala and evaluate antibacterial activities of the isolated compounds. Crude gradient extracts were obtained from five solvents (petroleum ether, chloroform, acetone, methanol and water) with increasing solvent polarity using cold maceration technique. The in vitro antibacterial activity evaluation of gradient extracts and isolated compounds was done on four different pathogenic bacterial strains (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Salmonella Typhimurium) using agar disc diffusion technique. Among the five crude extracts evaluated, the acetone extract was found to be the most active against the tested strains. Thus, this extract was subsequently subjected to column chromatographic separation which led to isolation of four compounds (MS-1, MS-2, MS-3 and MS-4). The chemical structures of the compounds were found to be cholest-5-en-3-ol, palmitic acid, n-octacosane and oleic acid, respectively, based on physical properties and spectroscopic (IR and NMR) data as well as literature reports. Three of the compounds namely cholest-5-en-3-ol, palmitic acid and oleic acid showed highest activity against E. coli. The observed antibacterial activities of the crude extract and the isolated compounds could justify the traditional use of the plant for the treatment of different bacterial infections. Thus, further test is recommended on large number of bacterial strains to decide their potential as candidates in development of antibacterial drugs.