In this study, a biosurfactant-producing bacterial strain was isolated from oil-contaminated soil on the basis of its ability to degrade crude oil and tributyrin ($C_{4:0}$). LSC04 was identified as Bacillus amyloliquefaciens LSC04 via 16S rRNA gene analysis and partial gyrA gene sequence analysis. The biosurfactants were purified and structural analysis results showed that B. amyloliquefaciens LSC04 generated a lipopeptide biosurfactant. Two main ions of 1,086.9 and 1,491.2 were measured via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The m/z 1,491.2 was shown to correspond to the lipopeptide fengycin B, but the m/z 1,086.9 ion did not correspond to any known lipopeptide. As constituents of the peptides and the lipophilic portion of the m/z 1,491.2;10 amino acids (Ile-Tyr-Gln-Pro-Val-Glu-Ser-Tyr-Orn-Glu); and ${eta}$-hydroxy-C17 fatty acid were identified via ESI-MS/MS. Structurally, the lipopeptide of a molecular mass of 1,491.2 differed from fengycin B and fengycin A by a substitution of serine for the threonine residue in position 4, and the amino acid residue in position 6 was equal to that of fengycin A. The major compound, which had a molecular mass of 1,491.2 Da was designated "Fengycin S".