The spectrum of antimicrobial effects of melittin were investigated on 19 pathogens by using a cylinder-plate method with serial dilutions. Bacteriostatic efficiency and possible mechanistic effects were monitored via growth curves. The mechanism of inhibition was further analyzed by SDS-PAGE, flow cytometry and electron microscopy. Melittin had a wide inhibition spectrum and killed pathogens effectively, and bacteriostatic action was influenced by factors such as pH and temperature. We elucidated three inhibitory mechanisms: melittin integrated with the cell membrane causing cell bursting and cytoplasm release, inhibited the synthesis of proteins and caused the cytoplasm to condense, and delayed pathogens in phase I (or phase G1) so that they could not complete the cell cycle. These results suggest that melittin could serve as a broad-spectrum biological pesticide with fast-action and high-efficiency.