Hospital-acquired infections caused by multidrug-resistant bacteria pose significant challenges for treatment, which necessitate the development of new antibiotics. Antimicrobial peptides are considered potential alternatives to conventional antibiotics. The skin of Anurans (frogs and toads) amphibians is an extraordinarily rich source of antimicrobial peptides. CPF-C1 is a typical cationic antimicrobial peptide that was originally isolated from the tetraploid frog Xenopus clivii. Our results showed that CPF-C1 has potent antimicrobial activity against both sensitive and multidrug-resistant bacteria. It disrupted the outer and inner membranes of bacterial cells. CPF-C1 induced both propidium iodide uptake into the bacterial cell and the leakage of calcein from large liposome vesicles, which suggests a mode of action that involves membrane disturbance. Scanning electron microscopy and transmission electron microscopy verified the morphologic changes of CPF-C1-treated bacterial cells and large liposome vesicles. The membrane-dependent mode of action signifies that the CPF-C1 peptide functions freely and without regard to conventional resistant mechanisms. Additionally, it is difficult for bacteria to develop resistance against CPF-C1 under this action mode. Other studies indicated that CPF-C1 had low cytotoxicity against mammalian cell. In conclusion, considering the increase in multidrug-resistant bacterial infections, CPF-C1 may offer a new strategy that can be considered a potential therapeutic agent for the treatment of diseases caused by multidrug-resistant bacteria. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.