Surfactin is a cyclic lipopeptide produced by Bacillus subtilis. This antibiotic disturbs the integrity of the membrane by forming pores and, with increasing concentration it completely disrupts the bilayer by a detergent mechanism. Due to amphiphilic nature surfactin solubility in water is compromised and therefore water solutions of alkaline pH are used (10 mM NaOH) for dissolution. Another widely used option is to dissolve surfactin in dimethyl sulfoxide (DMSO). In our study we investigated surfactin’s pore forming activity using black lipid membranes. We show that the pore-forming and permeabilizing activity of surfactin varies (i) for the different surfactin stock solutions used (DMSO vs. water) and (ii) for different phospholipid compositions of the target bilayer. Surfactin at the concentration of 2 µM in water formed variety of pores with broad conductance distribution ranging from few pS to over 1 nS (DPhPG; 1 M KCl, 10 mM Tris, pH 7.4) with Poisson-like distribution. At higher surfactin concentration (4 µM) the most frequent conductance unit of 10 pS remained, however, the occurrence of pores with higher conductance increased. In contrast, when surfactin DMSO solution was applied the membrane conductivity increased gradually and no individual pore openings were observed. Liposome leakage of carboxyfluorescein loaded vesicles of different phospholipid compositions showed that the susceptibility to surfactin-induced leakage as well as the mechanism of liposome lysis is strongly dependent on the composition of the target bilayer. Again, the kinetics of liposome leakage differed for the two surfactin stock solution used, surfactin in DMSO being more potent membrane permeabilizer. In both cases acidic phospholipids with higher melting temperatures such as cardiolipin and phosphatidic acid attenuated the membrane-perturbing effect of surfactin. We propose that the different mode of surfactin action in the two stock solutions may stem from different miscibility and aggregation of surfactin within membrane phospholipids.