Abstract: In order to investigate the antibacterial effect and mechanism of extracts fermented by Bacillus subtilis, the minimum inhibitory concentration (MIC) of the extracts on E.coli was analyzed by double dilution method. The growth curve, cell membrane permeability and cell ultrastructure were employed to investigate the antibacterial mechanism of the extracts on E.coli. Meanwhile, the compounds types of the extracts were analyzed preliminarily. The results showed that the extracts had a significant inhibitory effect on the growth of E. coli, and its MIC value was 0.614 mg/mL. In addition, the extracts added in the log phase and lag phase had better effect than that in the stable phase during the growth of E.coli. The extracts could lead to the roughness in cell surface and the rupture of cell membrane, and increase the cell membrane permeability. The compounds analysis showed that the main antibacterial components in the extracts were polyene and lipopeptide compounds.