Enterocin CRL35 activity is deeply influenced by the membrane viscosity as could be demonstrated performing determinations of the minimal inhibitory concentrations (MIC) at different temperatures and analyzing the membrane viscosity in these cells as well as in resistant bacteria. In all the cases, bacteriocin activity was linked to higher levels of viscosity. This finding was confirmed studying the interaction of enterocin CRL35 with liposomes composed of dimyristoyl phosphatidylcholine: dimyristoyl phosphatidylglycerol (9:1) in both gel and liquid-crystalline phases. It could be establish, from peptide insertion analysis following the tryptophan fluorescence and microviscosity experiments that this peptide is able to interact more efficiently with membranes having a more structured hydrophobic core.