Surfactin is a lipopeptide antibiotic produced by Bacillus subtilis. It acts by permeabilizing target cell membrane leading to disruption of membrane potential, cell content leakage or even membrane disintegration. Molecular details of this action have not yet been fully explained, nor has the producer’s mechanism of resistance. Typical resistance mechanisms such as single point mutation cannot be applied here as the target is the whole membrane structure and its barrier function rather than a single protein in a single metabolic pathway. And indeed, no resistance protein or gene was identified to be indispensable for surfactin resistance to date. Complex target-structure modification seems to be the most probable mechanism. We have previously shown that B. subtilis drastically alters its membrane lipid composition upon surfactin exposure, mainly increasing the content of cardiolipin. In this study, we further examine the effect of different phospholipids on membrane sensitivity to surfactin induced permeabilization on model membrane systems. We employ both the membranes composed of pure phospholipids and lipids isolated from B. subtilis wild type and cardiolipin defective strains. Our results show that surfactin activity is dependent on target membrane composition. Higher cardiolipin content protects the membrane against permeabilization by surfactin and thus can indeed be the major part of mechanism B. subtilis employs to protect itself against its own toxic product.