Bacillus subtilis N-2 which was isolated from natto, produced lipopeptides using soybean meal as a substrate. This work aimed to purify, identify, and determine the antibacterial mechanism of lipopeptides produced by B. subtilis N-2. The fermented product obtained by solid-state fermentation was subjected to water extraction, acid precipitation, and methanol extraction. Fractions were separated and collected using a two-step ultrafiltration method and then identified by LC–MS/MS. Mass spectrometry characterization revealed the presence of four variants of iturin A that differed according to the β-amino fatty acid chain from C 14 to C 17 as well as the amino acid positions. A new lipopeptide (m/z 1070.3) was identified and its structure was different from the previously reported lipopeptides. The lipopeptides were shown to inhibit the growth of an isolate of Pseudomonas putida, a common pathogen in decaying fish, by changing membrane permeability. These results suggest that the lipopeptides from B. subtilis N-2 could be used as a biocontrol agent in aquaculture.