Foodborne bacteria, especially from fermented foods, are ideal sources for probiotics because they are safe, gut-adapted, and capable of inhibiting harmful microbes while supporting human health. In this work, we demonstrated that Bacillus siamensis strain SNU247 (BS247), isolated from fermented soybean paste, inhibited the growth of bacterial pathogens such as Pseudomonas aeruginosa (PA14) and Staphylococcus aureus (SA3) in vitro. To genetically assess the antibacterial activity, we sought to identify the gene(s) with an ethyl methane sulfonate (EMS)-induced mutation affecting the in vitro antibacterial activity against PA14 and SA3. Three mutants (2D9, 5C9, and 19B3) were isolated from 2,016 EMS mutants but displayed different antibacterial characteristics: 2D9 and 5C9 were compromised in the killing activity against PA14, whereas 19B3 was impaired in that against SA3. The topics discussed will cover the genomic profile of BS247 and the genetic characterization of the isolated mutants based on whole genome sequence comparison.