Aims: In a search for an antifungal peptide with a high activity against Aspergillus flavus, Bacillus subtilis AU195 was selected from a collection of isolates with antagonistic activity against A. flavus. Methods and Results: To identify the antifungal peptides, a protein purification scheme was developed based on the detection of the antifungal activity in purified fractions against A. flavus. Two Lipopeptides were purified with anion exchange and gel filtration chromatography, Their masses were determined to be 1045 and 1059 m/z with mass spectrometry, and their peptide moiety was identical to bacillomycin D. Conclusions: AU195 synthesized a mixture of two antifungal bacillomycin D analogues with masses of 1045 and 1059, the 14 mass unit difference representing the difference between a C15 and a C16 lipid chain. Significance and Impact of the Study: Both bacillomycin D analogues were active at the same concentration against A. flavus, but the different lipid chain length apparently affected the activity of the lipopeptide against other fungi.