Prodigiosin is a natural red pigment from a family of prodiginins. It is interesting because of its antibacterial, antifungal, algicidal and antiprotozoal activity. In addition, the anticancer and immunosuppressive properties of prodigiosin were described. The mechanism of prodigiosin's antibacterial action is still unknown. The aim of the study was to determine cellular target components which prodigiosin affects and causes bacterial cell lysis. Minimal inhibitory concentration (MIC) of Bacillus sp. was evaluated by using broth dilution method in microtiter plates. MIC was also evaluated at different incubation temperatures and different medium salinity. The antibacterial activity was evaluated with measuring optical density of bacterial strains in the LB medium at 650 nm, where prodigiosin was added at different cell growth phases. For cell viability evaluation differential interference contrast microscopy was used and cytoplasmic membrane leakage was determined by measuring β – galactosidase activity. As a control, B. subtilis PS-216 was treated with different bacteriolytic and bacteriostatic agents. We have demonstrated, that all tested Bacillus strains are susceptible to prodigiosin. MIC value was between 2,82 ± 1,81 mg/L against Bacillus sp. 5A and 7,83 ± 2,86 mg/L against B. subtilis NCIB 3610. Exposure of bacterial strains to stress caused by temperature fluctuation and NaCl concentraton increased susceptibility to prodigiosin. Microscopy and optical density data indicate that prodigiosin causes celly lysis. The highest impact of prodigiosin was observed on actively dividing cells in the exponental growth phase. We have observed that prodigiosin increases permeability of cytoplasmic membrane. Based on our results, we have proposed that prodigiosin induces autolysins, leading to fast autolysis of bacterial culture.