Search for novel antimicrobial agents, including plant-derived flavonoids, and evaluation of the mechanisms of their antibacterial activities are pivotal objectives. The goal of this study was to compare the antihemolytic activity of flavonoids, quercetin, naringenin and catechin against sheep erythrocyte lysis induced by α-hemolysin (αHL) produced by the Staphylococcus aureus strain NCTC 5655. We also sought to investigate the membrane-modifying action of the flavonoids. Lipophilic quercetin, but not naringenin or catechin, effectively inhibited the hemolytic activity of αHL at concentrations (IC₅₀ = 65 ± 5 µM) below minimal inhibitory concentration values for S. aureus growth. Quercetin increased the registered bacterial cell diameter, enhanced the fluidity of the inner and surface regions of bacterial cell membranes and raised the rigidity of the hydrophobic region and the fluidity of the surface region of erythrocyte membranes. Our findings provide evidence that the antibacterial activities of the flavonoids resulted from a disorder in the structural organization of bacterial cell membranes, and the antihemolytic effect of quercetin was related to the effect of the flavonoid on the organization of the erythrocyte membrane, which, in turn, increases the resistance of the target cells (erythrocytes) to αHL and inhibits αHL-induced osmotic hemolysis due to prevention of toxin incorporation into the target membrane. We confirmed that cell membrane disorder could be one of the direct modes of antibacterial action of the flavonoids.