With the global warming, the cultivation and storage of northern Chinese cabbage has been infected by Sclerotium bacteria, resulting in the whole plant rot. In this study, in order to develop a bio-inhibitory agent with antibacterial effect and no toxicity and no residue, three strains with high inhibition rate against Sclerotinia sclerotiorum were screened from 69 samples of Doenjang collected in different regions. By observing culture characteristics and physiological and biochemical tests, three strains were identifi ed through 16S rDNA sequence and phylogenetic tree. The results showed that SP-6 was Bacillus amyloliquefaciens, SP-13 was Bacillus methylotrophicus, and SP-14 was Bacillus subtilis. Meanwhile, the inhibition rate of the aseptic fermentation fi ltrate of three strains against S. sclerotiorum was determined and the fermentation conditions of SP-14 strain was optimized. Results showed that all of three fermentation filtrates inhibited S. sclerotiorum. Data showed that the best conditions for fermentation for the antibacterial substances of SP-14 strain is as following: 1% maltose, 1% ammonium oxalate or beef extract, 1% ferrous sulfate, fermentation at 32 °C for 36 h, initial pH 8–9 and the inoculation amount 5%. The living bacteriostatic test showed that SP-14 strain had good inhibitory effect on S. sclerotiorum and the inhibitory effect was more signifi cant when the bacteria concentration was 1.2 × 1011 CFU/mL.