A novel antimicrobial peptide named Pep39 has been isolated from anchovy hydrolysate using ÄKTA protein liquid chromatography and reverse-phase high-performance liquid chromatography. Its amino acid sequence was RLFRHAFKAVLRL with a molecular weight of 1626.92. Pep39 demonstrated antimicrobial ability against Escherichia coli ATCC 25922, Salmonella typhi ATCC 50013, Staphylococcus aureus ATCC 25923 and Bacillus subtilis ATCC 9372, with minimal inhibitory concentration (MIC) ranging from 4 to 32 μg mL−1. No cytotoxicity to mouse red blood cells was observed when its concentration was lower than 30 μg mL−1. Pep39 induced the influx of fluorescent probe 8-Anilino-1-naphthalenesulfonic acid and the outflow of β-galactosidase by increasing E. coli outer and inner membrane permeabilities, respectively. Flow cytometric analysis also demonstrated that Pep39 disrupted E. coli cell membrane. These results suggested that the antimicrobial mechanism of Pep39 involved cytoplasmic membrane damage.