[Objective] To isolate and identify lipopeptides in metabolites of Bacillus subtilis subsp. natto Bna05 and to assess antifungal activity of the identified lipopeptides. [Methods] Primers specific for lipopeptide synthetases were used to amplify DNA fragments from strain Bna05 by PCR. The amplified DNA fragments were sequenced and analyzed using BLAST against GenBank database. The antifungal metabolites were obtained from the inhibition zone formed between Bna05 and Aspergillus niger on modified PDA plate and separated by RP-HPLC. Antifungal activity of fractions separated by RP-HPLC was determined by micro-dilution method and the active molecules were identified by mass spectrum analysis. [Results] Genes for srfAA and sfp were identified in strain Bna05. However, genes encoding for ituC, ituD, fenD, fenACE, bymB and bymC were not detected. Three active fractions F2, F3 and F4 were isolated by RP-HPLC. Lipopeptide was not detected in F2. Two kinds of surfactin variants were identified from F3 and F4: V7-surfactin variants in F3 and I/L7-surfactin variants in F4. Synergistic effect against Aspergillus niger was observed when F2 was used together with either V7-surfactins or I/L7-surfactins. The best antifungal activity was obtained when all three fractions were used together. [Conclusion] V7-surfactins and I/L7-surfactins were the main lipopeptides secreted by B. subtilis natto Bna05. Synergistic antifungal effect was found against Aspergillus niger when surfactins were used together with other active compounds such as F2. The greatest synergistic effect was obtained when both V7-surfactins and I/L7-surfactins were used together with F2.