Maggots (larvae of Lucilia sericata) have shown therapeutic effects on refractory wounds infected with bacteria, yet the bacterial killing mechanisms are unclear. Herein, we report the isolation and purification of an antibacterial protein from maggots (MAMP). MAMP demonstrated inhibitory activity against both standard strains and clinically isolated antibiotic-resistant strains of Staphylococcus aureus in vitro. The topical use of MAMP effectively decreased the viability of S. aureus and promoted wound healing in an S. aureus mouse skin infection model. MAMP exerted its antibacterial activity via a bactericidal mechanism based on observations using scanning electron and transmission electron microscopy. MAMP interacted with the bacterial cell membrane and disrupted the cell surface structure. In addition, MAMP had weak hemolytic activity at a high concentration. Taken together, MAMP exhibits potential use as a topical agent for treating bacterial infections.