Traditional Chinese fermented foods, known for their probiotic content and microbial diversity, contribute significantly to dietary nutrition. This study aimed to assess the safety and antimicrobial potential of Bacillus isolated from these foods for industrial applications. Evaluating the antimicrobial activity and safety of probiotics such as Bacillus is imperative for application in the fermented food industry. Sixty Bacillus strains were isolated from 60 fermented food samples and confirmed by 16S rDNA testing. Of these, 39 were identified as non-toxic based on PCR screening, suggesting a favorable safety profile for consumption. Among the pathogens tested, B. subtilis YH91-2 demonstrated the most potent antibacterial activity against both Gram-positive (n = 2) and Gram-negative (n = 7) bacteria. Comprehensive analyses of hemolytic evaluation, DNA enzyme activity, antibiotic susceptibility, cytotoxicity tests, and tolerance to unfavorable conditions, B. subtilis YH91-2 had the potential as a promising probiotic. Besides vitro evaluation, the effects of B. subtilis YH91-2 in vivo safety were also examined. No harmful health effects were observed in mice irrigating with high, medium, and low doses of YH91-2 for 21 days. Notably, mice receiving high doses showed significant weight gain and a marked decrease in triglyceride levels compared to control subjects. The results provided compelling evidence for the potential of B. subtilis YH91-2 as a probiotic for advancing food industry applications.