Abstract In recent years, the use of microorganisms to stimulate plant growth and resistance, including biocontrol and maintenance or improvement of microbial activity and soil functionality, has increased. The aim of this work was to select nutrient media and to optimize the deep cultivation regimes of a biopreparation consisting of the Bacillus pumilus Pol P3(1) 10 B-RKM 0528, Bacillus thuringiensis Pb 30 B-RKM 0341, Bacillus licheniformis 356 B-RKM 0074 and Serratia marcescens Sh-2 B-RKM 0952 strains, which exhibit high growth-stimulating activity. Three different media were use to cultivate the biopreparation: standard meat-peptone broth medium, King's medium and Mueller-Hinton's medium. The main criterion for determining the effectiveness of the nutrient medium was the accumulation of specific components to yield the largest biomass of biopreparation cells. A comparative analysis of growth on the nutrient media was used to assess the viability and biomass accumulation of the biopreparation strains tested. The optimum cultivation period, maximum optical density of biomass growth, viability and optimum medium acidity during bacterial cultivation on nutrient media were determined both for the strains individually and as part of the biopreparation.