Abstract: A new strain with broad-spectrum anti-Bacillus spp. activity, JLY-7, was isolated from Korean traditional fermented kimchi samples. The cell-free supernatant produced by the strain JLY-7 showed a high inhibitory activity towards common pathogenic and food-spoiling Bacillus spp. such as Bacillus cereus, Bacillus subtilis, Bacillus coagulans, Bacillus megaterium, Bacillus pumilus, Geobacillus stearothermophilus and Paenibacillus polymyxa. The strain JLY-7 was identified as Lactobacillus plantarum by morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis. Then, the antimicrobial substance was isolated and purified using butanol extraction, gel filtration chromatography (Sephadex LH-20) and semi preparative HPLC and a single antimicrobial component was obtained. The molecular mass of the purified antimicrobial substance was estimated to be 694.129 Da by HR-ESI-MS. Protease treatment results showed that the antimicrobial substance was sensitive to all proteases. The research was conducted to lay a theoretical foundation for controlling Bacillus spp. in food by antimicrobial substance produced from lactic acid bacteria.