Herein, a bactericide-secreting Bacillus strain, potentially useful as a biocontrol agent, was isolated from the commercial Yanjing Natto food. Following the biochemical and physiological evaluation, the molecular identification was performed using 16S rDNA sequencing of polymerase chain reaction-amplified DNA that confirmed the natto isolate as Bacillus subtilis natto (B. subtilis natto). The biocontrol (microbial inhibitory) capability of B. subtilis natto was investigated against Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, and yeast (Yarrowia lipolytica) and recorded. The antimicrobial activity of B. subtilis natto was further enhanced by optimizing the growth medium for optimal bactericides secretion. Under optimized conditions, B. subtilis natto exhibited much higher inhibitory activity against S. aureus with a zone of inhibition diameter up to 27 mm. After 48 h incubation, the optimally yielded B. subtilis natto broth was used to extract and purify the responsible bactericides by silica gel column chromatography, gel column chromatography, and semi-preparative high-performance liquid chromatography. Structural identification of purified bactericides (designated as NT-5, NT-6, and NT-7) from B. subtilis natto was performed by 13C-nuclear magnetic resonance (NMR) and mass spectral analyses. The NMR comparison also revealed that NT-5, NT-6, and NT-7 had identical structures, except for the fatty chain. In summary, the present study suggests the improved biocontrol and/or microbial inhibitory potential of newly isolated bactericides secreting B. subtilis natto.