The invention relates to the field of biotechnology, in particular to a method for separating and extracting bacillus subtilis lipopeptid antibacterial material. The method more particularly comprises the steps Sephadex G-25 column chromatography is carried out on crude extract of fermentation liquor of the bacillus subtilis, active peak is collected by chromatography, and the Sephadex G-25 column chromatography is carried out on the active peak which is collected by Sephadex G-25 column; the active peak is collected, and the obtained substance is seven lipopeptid antibacterial materials after being detected by mass spectrum. Furthermore, the extracted lipopeptid antibacterial material has good inhibition effect on multiple pathogenic fungis, and has the advantages of stable temperature and pH value, prolease hydrolysis resistance, etc.