This study investigates the antibacterial action of chitosan-based nanofibers (CNFs) obtained by the electrospinning process on the permeability of bacterial membranes. The bactericidal efficiency of CNFs was first determined against Gram-negative Escherichiacoli and Salmonella Typhimurium, and Gram-positive Staphylococcusaureus and Listeriainnocua bacteria as a baseline. The results strongly suggest that CNFs interact with the negatively charged bacterial cell wall causing membrane rupture and inducing leakage of intracellular components among which are proteins and DNA. Results clearly indicate that the release of such components after contact with CNFs is an indication of membrane permeabilization and perforation, as pore formation was observed in transmission electron microscopy (TEM). This work suggests a plausible antibacterial mechanism of action of CNFs and also provides clear evidence in favor of chitosan as a bacterial membrane disruptor and perforator. As a result, CNFs can find promising applications as bioactive food packaging materials capable to extend shelf life of food products while inhibiting the spread of alteration flora and foodborne pathogens.