Optimization of the cultivation medium for production of antibiotic effective against pathogenic bacteria Staphylococcus aureus using strain of Streptomyces spp. isolated from the environment represents the aim of this study. After the biosynthesis, the medium was analyzed by determining residual sugar and nitrogen, and the antibiotic activity was determined using diffusion-disc method. Experiments were carried out in accordance with the Box-Behnken design, with three factors varied on three levels (glucose: 10.0, 30.0 and 50.0 g/L; soybean meal: 5.0, 15.0 and 25.0 g/L; phosphates: 0.5, 1.0 and 1.5 g/L) and for the optimization of selected parameters Response Surface Methodology was used. The obtained model with the desirability function of 0.985 estimates that the lowest amounts of residual sugar (0.89 g/L) and nitrogen (0.24 g/L) and the largest possible inhibition zone diameter (21.88 mm) that with its antibiotic activity against S. aureus creates the medium containing 10.0 g/L glucose, 5.0 g/L soybean meal and 1.04 g/L phosphates.