Biosurfactants are microbial surface active compounds which, contrary to synthetic surfactants, are natural in origin, biodegradable and less toxic to a human organism. For that reason, there is a great research potential in studies aimed at their purification, finding potential ways of their utilization and decreasing their production costs. This paper demonstrates the process of isolating and purifying a surfactin synthesized by Bacillus subtilis PCM 1949. Surfactin samples were prepared by a classical organic solvent extraction and were studied using mass spectrometry (MS). Analysis of the susceptibility profile of microorganisms utilized in the diffusion-plate tests demonstrated that their sensitivity to this surfactin is differentiated and depends on the microorganism species. In our studies, we found that the selected strains of bacteria and fungi were insensitive to this surfactin at a wide range of concentrations.