Recently several insects have been identified as potential carriers of antimicrobial peptides (AMPs), which have potent activity against pathogens. Wasp venom AMPs serve as defense agents against invading microorganisms.AMPs derived from wasp venom have high attention as therapeutic agents against infectious agents with novel mechanisms of action. In the present study, the antibacterial activity of the oriental hornet “Vespa orientalis L. (Hymenoptera: Vespidae)" venom was determined against three Gram-positive and three Gram-negative bacteria; and the minimum concentration of the venom that inhibits the bacterial growth was determined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique was used for fractionation of the V. orientalis venom peptides. The effect of V. orientalis AMPs on bacterial cell membrane and cytoplasm was also investigated; destruction of bacterial cell wall inhibited steps of the synthesis of important molecules and caused cell death. The results proved the highly effective antibacterial properties of the V. orientalis venom. Therefore, peptides of the V. orientalis venom could be potential alternative agents to the currently-used antibiotics.