Snake venom phospholipase A2 (PLA2) has widely been reported to possess antibacterial effects, and PLA2 is the major component of cottonmouth snake venoms. We assessed the antibacterial activities of crude venoms from Western cottonmouth (Agkistrodon piscivorus leucostoma), Eastern cottonmouth (Agkistrodon piscivorus piscivorus), and Florida cottonmouth (Agkistrodon piscivorus conanti) snakes against two gram-positive (Bacillus subtilis, Staphylococcus aureus), and two gram-negative (Escherichia coli, Vibrio cholerae) bacteria. Antibacterial activity of PLA2 proteins, AplAsp49 and AplLys49 purified from A. p. leucostoma venom, was also examined. Disk-diffusion assays revealed that A. p. leucostoma crude venom is most effective in inhibiting the growth of the bacteria tested, compared to the other two. Surprisingly, AplAsp49 and AplLys49 PLA2s purified from A. p. leucostoma venom did not display detectable antibacterial activity against any bacteria tested neither by disk-diffusion nor by minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). The lack of antibacterial activity of cottonmouth venom PLA2s is discussed.