The present work focuses on one of the possible target mechanisms of action of the anionic antimicrobial peptide β-lg125-135 derived from trypsin hydrolysis of β-lactoglobulin. After confirmation of bactericidal activity against a pathogenic Gram(+) strain and demonstration of the innocuousness on a eukaryotic cell line, we investigated the interaction of β-lg125-135 with monolayers and bilayers of dpPC and dpPC:dpPG as model membranes of eukaryotic and bacterial membranes, respectively. In monolayers, compared to zwitterionic dpPC, in the negatively charged dpPC-dpPG, β-lg125-135 injected into the subphase penetrated up to higher surface pressures and showed greater extents of penetration with increasing concentration in the subphase. Additionally, the rate constants for β-lg125-135 adsorption and desorption were 1 order of magnitude higher, and the resultant thermodynamic association constant was 1 order of magnitude lower. In turn, the compression isotherms of monolayers prepared with the β-lg125-135 present in the mixture spread over the air-water interface, remained in the monolayer and showed positive deviations from ideality, a greater decrease in the surface compressibility modulus, and an increase in the surface potential of both interfaces, more pronounced on dpPC:dpPG. In SUVs, fluorescence anisotropy (FA) assays using DPH and TMA-DPM indicated that β-lg125-135 tended to disrupt the gel phase of dpPC bilayers. Conversely, in dpPC:dpPG, the peptide increased the FA of both probes. These results reflect a relatively high tendency of the β-lg125-135 to approach the negative interface, with a favorable electrostatic orientation but low stability and short residence time. Once inside the membrane, it stiffens dpPG-containing bilayers.