Cathelicidin-BF (BF-30) is found in the venom of the snake Bungarus fasciatus and exhibits broad antimicrobial activity against bacteria and fungi. Nevertheless, its antibacterial activity in vivo and antibacterial mechanism is unknown. In the present study, we examined the antibacterial activity of BF-30 in vitro against drug-resistant Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus, first identifying its protection against P. aeruginosa in infected burns and then delineating the antimicrobial mechanism of BF-30. The data showed that BF-30 had stronger antimicrobial activities against a broad spectrum of microorganisms than gentamicin, ampicillin or bacitracin. The killing curves of BF-30 against P. aeruginosa and S. aureus showed that CFU counts rapidly decreased by almost 2 logs within 6min, and it took just less than 2h to kill all the bacteria. In addition, we investigated whether BF-30 had antibacterial activity in a burn/acute infection rat model. Dose-response (0.75, 3, 12mg/kg/day) studies indicated that BF-30 significantly reduced the colonization of P. aeruginosa in the burn eschars, lungs and liver of burn injured rats and that it could prevent subsequent systemic infection and development of inflammation. The peptide induced chaotic membrane morphology and cell debris, as determined by electron microscopy, and caused the cytoplasmic membrane to crack, resulting in β-galactosidase leakage and EtBr accumulation. This suggests that the antimicrobial activity of BF-30 is based on cytoplasmic membrane permeability. Taken together, our data demonstrate that antibacterial activity of BF-30 has potential therapeutic value for the prevention and treatment of burn and wound infections.