This study investigates whether the B chain of β-bungarotoxin exerted antibacterial activity against Escherichia coli (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria) via its membrane-damaging activity. The B chain exhibited a growth inhibition effect on E. coli but did not show a bactericidal effect on S. aureus. The B-chain bactericidal action on E. coli positively correlated with an increase in membrane permeability in the bacterial cells. Lipopolysaccharide (LPS) layer destabilization and lipoteichoic acid (LTA) biosynthesis inhibition in the cell wall increased the B-chain bactericidal effect on E. coli and S. aureus. The B chain induced leakage and fusion in E. coli and S. aureus membrane-mimicking liposomes. Compared with LPS, LTA notably suppressed the membrane-damaging activity and fusogenicity of the B chain. The B chain showed similar binding affinity with LPS and LTA, whereas LPS and LTA binding differently induced B-chain conformational change as evidenced by the circular dichroism spectra. Taken together, our data indicate that the antibacterial action of the B chain is related to its ability to induce membrane permeability and suggest that the LPS-induced and LTA-induced B-chain conformational change differently affects the bactericidal action of the B chain. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.