Antifungal metabolites produced by Bacillus sp. W10, which was previously isolated from the tomato rhizosphere, were investigated. Strain W10 was identified as Bacillus amyloliquefaciens by analysis of its 16S rDNA and gyrB gene partial sequences. PCR analysis showed the presence of fenB, sfp, and ituD genes, coding for fengycin, surfactin, and iturin, respectively. A novel small antifungal peptide, designated 5240, produced by this strain was isolated by ammonium sulfate precipitation and Superdex 200 gel filtration chromatography. The 5240 peptide was stable at 100 °C for 20 min and remained active throughout a wide pH range (4-10). The antagonistic activity was not affected by protease K and trypsin. The purified 5240 peptide exhibited a broad inhibitory spectrum against various plant pathogenic fungi and was identified as iturin A (C-C). Moreover, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry indicated the presence of fengycin A (C-C), fengycin B (C-C), and surfactin (C-C) isoforms in supernatants from strain W10. These results suggest that B. amyloliquefaciens W10 has significant potential as a biocontrol agent.