A Bacillus strain, denoted as PY-1, was isolated from the vascular bundle of cotton. Biochemical, physiological and 16S rDNA sequence analysis proved that it should belong to Bacillus subtilis. The PY-1 strain showed strong ability against many common plant fungal pathogens in vitro. The antibiotics produced by this strain were stable in neutral and basic conditions, and not sensitive to high temperature. From the culture broth of PY-1 strain, five antifungal compounds were isolated by acidic precipitation, methanol extraction, gel filtration and reverse-phase HPLC. Advanced identification was performed by mass spectrometry and nuclear magnetic resonance spectroscopy. These five antifungal compounds were proved to be the isomers of iturin A: A2, A3, A4, A6 and A7. In fast atom bombardment mass spectrometry/mass spectrometry collision-induced dissociation spectra, fragmentation ions from two prior linear acylium ions were observed, and the prior ion, Tyr-Asn-Gln-Pro-Asn-Ser-betaAA-Asn-CO+, was first reported.