In the effort to overcome the increase in antimicrobial resistance of different pathogens, natural products from microbial sources appear to be the most favorable alternative to current antibiotics. Production of antimicrobial compounds is highly dependent on the nutritional conditions. Hence, in order to achieve high product yields, selection of the media constituents and optimization of their concentrations are required. In this research, the possibility of antimicrobial substances production using Bacillus subtilis ATCC 6633 was investigated. Also, optimization of the cultivation medium composition in terms of contents of glycerol, sodium nitrite and phosphates was done. Response surface methodology and the method of desirability function were applied for determination of optimal values of the examined factors. The developed model predicts that the maximum inhibition zone diameters for Bacillus cereus ATCC 10876 (33.50 mm) and Pseudomonas aeruginosa ATCC 27853 (12.00 mm) are achieved when the initial contents of glycerol, sodium nitrite and phosphates were 43.72 g/L, 1.93 g/L and 5.64 g/L, respectively. The results of these experiments suggest that further research should include the utilization of crude glycerol as a carbon source and optimization of composition of such media and cultivation conditions in order to improve production of antimicrobial substances using Bacillus subtilis ATCC 6633.