Since the ancient times, the antibacterial application of honeybee venom (BV) has been practised and persisted. We investigated the antibacterial activity of whole BV and purified melittin against Escherichia coli and Staphylococcus aureus by the minimum inhibitory concentrations (MICs) and the postantibiotic effects (PAEs). The in vitro PAEs of whole BV and isolated melittin were determined using E. coli and S. aureus. The PAEs of whole BV against E. coli and S. aureus were 0.15 h (1 MIC), 2.4 h (5 MIC), and 3.45 h (1 MIC), respectively. The PAEs of melittin against E. coli and S. aureus were 0.1 h (1 MIC), 3.2 h (5 MIC), and 4.35 h (1 MIC), respectively. These results suggest that whole BV and melittin will be developed as a novel antibacterial drug.