Abstract: In order to identify the antagonistic materials produced by Bacillus subtilis ge25, we used column chromatography and activity tracking methods to isolate and purify the lipopeptides from fermentation broth, and UPLC-ESI-Q-TOF to understand their chemical structure. Results indicated that, in total, 28 components were isolated by column chromatography. Components G23 to G27 were of antagonistic activity, and G25 and G26 expressed the highest. Three components, L1, L2 and L3 were identified from G25 and G26 by Silica gel column chromatography and thin-layer chromatography (TLC). Finally, analytic results showed that, one fengycin A and six fengycin B homologues or their derivatives from L2 and L3, two kinds of unknown chemicals from L1 were identified by UPLC-ESI-Q-TOF. As that the molecular weight and debris pyrolysis were different from known lipopeptides, and no clear amino acid sequence could be retrieval, we deduced that two lipopeptides in L1 were probably different from antagonistic lipopeptides reported. Scanning electron microscopy results indicated that antagonistic lipopeptides secreted by B. subtilis ge25 significantly led to abnormal mycelia growth of ginseng pathogens Alternaria panax and Cylindrocarpon destructans.