Multidrug resistance in foodborne and clinical pathogens is a worldwide health problem. The urgent need for new alternatives to the existing antibiotics is emerging. Bacteriocin-like inhibitory substances can be considered part of the new generation of antimicrobials, which can be potentially applied in the food industry and health care practices. This study aimed to select Bacillus strains with antimicrobial activity against Staphylococcus spp. with future application in the formulation of pharmaceutical antimicrobial preparations. Putative antimicrobial agent-producing strains, previously isolated and preidentified as Bacillus spp. were profiled by repetitive element sequence-based polymerase chain reaction (rep-PCR) and 16s rRNA sequencing identified the strains as Bacillus tequilensis ST1962CD with 99.47% identity confidence and as Bacillus subtilis subsp. stercoris ST2056CD with 98.45% identity confidence. Both the selected Bacillus strains were evaluated via biomolecular and physiological approaches related to their safety and virulence, beneficial properties, enzyme production profile, and presence of corresponding genes for the production of antimicrobials and virulence. Both strains were confirmed to harbor srfa and sbo genes and be free of hemolysin binding component (B) and two lytic components (L1 and L2) [BL] and nonhemolytic enterotoxin-associated genes. Produced antimicrobial agents by strains ST1962CD and ST2056CD were partially purified through the combination of ammonium sulfate precipitation and hydrophobic-based chromatography on SepPakC18 and evaluated regarding their cytotoxicity. The dynamics of bacterial growth, pH change, accumulation of produced antimicrobials, and the mode of action were evaluated. Obtained results were pointing to the potential application of safe B. tequilensis ST1962CD and B. subtilis subsp. stercoris ST2056CD strains as functional beneficial microbial cultures that are putative producers of surfactin and/or subtilosin, as potent antimicrobials, for the treatment of some staphylococcal-associated infections. Expressed antimicrobials were shown to be not cytotoxic, and appropriate biotechnological approaches need to be developed for cost-effective production, isolation, and purification of expressed antimicrobials by studied strains.