The purpose of these investigations was to determine the effect of environmental factors (nutrient concentrations, temperature, pH and time of culture) on surfactin and iturin biosynthesis by Bacillus spp. The studies were carried out with nine Bacillus spp. strains (also isolated from natural environments). To compare the effect of each factor on surface-active compounds production, the experiments were designed as a factorial search with the three levels for each variable and response surface method was used. The ability to lipopeptide biosurfactants production by examined Bacillus spp. strains was investigated using HPLC technique. Experiment design and results analysis were done by DoE programme. In the first step of experiments production of surfactin and iturin in optimal conditions was investigated. It has been shown that the yield of biosynthesis of lipopetide biosurfactants by examined Bacillus spp. strains varied according to species of bacteria. The highest production of surfactin was observed for Bacillus cereus (70,85 mg/l). Iturin biosynthesis was very poor in these conditions (from 0,01 to 0,35 mg/l). In the second step of investigations an influence of environmental factors on lipopetide biosurfactants was investigated. The target of this tests were of four different Bacillus spp. strains, the most effective towards to lipopetide production for whom culturing was done in designed conditions according to DoE model. Conducted experiments indicate that availability of nitrogen source, pH, interactions between pH in culture medium and content of glucose, influenced the surfactin production in all used strains. Statistically significant correlations were also noted between examined factors.