An antimicrobial activity produced by Bacillus subtilis B38 was found to be effective against several bacteria, including pathogenic and spoilage microorganisms such as, Listeria monocytogenes, Salmonella enteridis, and clinical isolates of methicillin-resistant Staphylococcus species. Nutrients such as carbon, nitrogen sources, and inorganic salts enhanced the production level of the antibacterial activity by B. subtilis B38. A first screening step showed that lactose, ammonium succinate, and manganese most influenced both cell growth and antibacterial activity production. These three factors varied at two levels in eight experiments using full factorial design. Results indicated that maximum cell growth (OD = 10.2) and maximum production of antibacterial activity (360 AU/mL) were obtained in a modified medium containing 1.5% (w/v) lactose, 0.15% (w/v) ammonium succinate, and 0.3 mg/L manganese. Depending on the indicator strain used, the antibacterial activity was 2- to 4-fold higher in the modified culture medium than in TSB medium under the same conditions. Thin layer chromatography-bioautography assay showed the presence of three active spots with R-f values of 0.47, 0.7, and 0.82 in TSB medium. However, the inhibition zone of two spots (Rf values of 0.7 and 0.82) was slightly larger in the modified medium. Moreover, a large zone of inhibition with an Rf value of 0.3, was observed in this modified medium, instead of the spot having an Rf value of 0.47. These results suggest that the nutrients act as environmental factors, quantitatively and qualitatively affecting the production of antibacterial compounds by B. subtilis B38. (c) 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 1267-1274, 2009