Bifidocin A exhibits strong activity against Listeria monocytogenes. The antibacterial mechanism of bifidocin A against L. monocytogenes was studied. Bifidocin A altered morphology and intracellular organization of L. monocytogenes. Bifidocin A increased the cell membrane permeability of L. monocytogenes. Bifidocin A resulted in pore formation in L. monocytogenes cell membranes.