Abstract: Antifungal activity of antimicrobial lipopeptide from Bacillus subtilis fmbJ to Penicillium notatum AS 3.4356 was studied to guide its application in the antisepsis of fruits. Antiseptic experiment of honey peaches was performed and antimicrobial spectrum was determined. The results indicate that Penicillium notatum is very sensitive to the lipopeptide and minimal inhibitory concentration (MIC) reaches 125 μg/mL. The lipopeptide has broad antimicrobial spectrum including gram positive bacteria, gram negative bacteria and fungi. It can strongly inhibit growth-breed and sporus germination of Penicillium notatum, and can lyse sporus of Penicillium notatum. It can reduce markedly activity of Succinate Dehydrogenase(SDH) and Malate Dehydrogenase(MDH) in Penicillium notatum, and can interfere or stop absorbing nutritional materials. Antiseptic experiment of peach demonstrated antiseptic rate of antimicrobial lipopeptide was 76.5％. So application prospect of antimicrobial lipopeptide from Bacillus subtilis fmbJ is better on antisepsis to peach.