The aim of the research was to study the features of the growth of B. subtilis CNMN-BB-09 bacteria on classical nutrient media and to determine the optimal composition of nutrient medium that promotes a high number of viable cells while maintaining antagonistic properties. Preliminary experiments showed that after two days of cultivation, the highest titer was observed on the medium MPB (4.1×109 CFU/ml), on the medium PD (1.2×109 CFU/ml), and on the medium M9 the titer was lower (3×108 CFU/ml). PD and MPB media are natural, whereas M9 medium is synthetic. Natural medium are not quite convenient to use for the development of culture, because of their high cost, and require the use of food and a long time for their preparation. Synthetic medium M9 is cheaper and easy to perform. Thus, the M9 medium can serve as a basis for further development of optimal medium for cultivation of bacteria B. subtilis CNMN-BB-09. The first stage of research was to determine the optimal source of carbon and nitrogen. The highest titers of 2.1 × 109, 5.9 × 109, 2.4 × 109 were reached for sucrose, maltose, glucose as sources of carbon, respectively. When we used sorbitol and dulcite as carbon sources, the growth of crop was inhibited. Ammonium and nitrate salts as well as glycine were used as sources of nitrogen nutrition in experiments. The best sources of nitrogen used in experiments are salts containing ammonium ions. The addition of some amino acids has affected the quantitative growth of studied bacteria. Thus, on the medium with threonine, methionine, lysine, valine, histidine, leucine and arginine, the culture titer after the first day of cultivation was in the range of 1.1 × 107 CFU/ ml – 5.3 × 107 CFU/ml. With the addition of tryptophan and phenylalanine, the culture titer reached only 9.8 × 106 CFU/ml and 6 × 106 CFU/ml, respectively. After two days of cultivation, titer of bacterial culture on the medium with lysine and histidine was the highest (1.6 × 109 CFU/ml). Slow growth of culture was observed on the medium with threonine and phenylalanine. After three days of cultivation, the highest titer of bacterial culture was on media with the addition of methionine, valine and tryptophan. After 4 days of cultivation, titer reached 1.2 × 1010 CFU/ml on the medium with arginine. Titer decreased sharply to 8.2 × 108 CFU/ml on the medium with leucine. When determining the effect of amino acids on antagonistic activity of B. subtilis CNMN-BB-09, it was observed that the addition of an amino acid to nutrient medium did not change the antifungal properties. In order to improve the growth characteristics and antifungal activity of culture, some additions were made into nutrient medium. These media are below numbers 23, 24, 25, 26, 27. The maximum titer of 2.6 × 109 CFU/ml for B. subtilis CNMN-BB-09 was observed on the nutrient medium M9 on day 4 of cultivation. Whereas, on the nutrient medium 27, titer was 7.5 × 109 CFU/ml after two days of cultivation, and on day 4 reached 1.7 × 1010 CFU/ml. The results of the experiment to determine the antifungal activity of tested bacterial strain cultivated on modified media are presented in Table 2. Analysis of the results showed that antibiotic substances were formed on the second day of culture growth. Thus, studies have shown that the optimal components of nutrient medium for cultivation of Bacillus subtilis CNMN-BB-09 are: dry yeast, sodium citrate, tryptone, glycerin. Therefore, the composition of optimized nutrient medium is as follows, (g/l): ammonium sulfate – 1.4, potassium phosphate single-substituted – 1.5, sodium phosphate dibasic – 2.5, sodium chloride – 0.5, glucose – 2.0, dry yeast – 4.0, sodium citrate – 0.25, trypton – 0.25, glycerin – 2.0 ml, water - the rest. The optimized nutrient medium is designed for growing Bacillus subtilis CNMN-BB-09 in the laboratory and makes it possible to obtain a significant yield of viable cells in 48 hours of cultivation. In addition, whenever cultivated on the proposed medium, high antagonistic properties of studied culture are preserved.