The aim of this project was to screen for bacteriocinogenic Bacillus strains with activity versus Staphylococcus spp. with future application in formulation of pharmaceutical antimicrobial preparations. Putative bacteriocinogenic strains, isolated and pre-identified as Bacillus spp. were selected for future study and differentiated based on repPCR and identified as Bacillus subtilis for strains ST826CD and ST829CD, Bacillus subtilis subsp. stercoris for strain ST794CD, Bacillus subtilis subsp. spizizenii for strain ST824CD, Bacillus velezensis for strain ST796CD, and Bacillus tequilensis for strain ST790CD. Selected strains were evaluated regarding their safety/virulence, beneficial properties, and potential production of antimicrobials based on biomolecular and physiological approves. Expressed bacteriocins were characterized regarding their proteinaceous nature, stability at different levels of pH, temperatures, and the presence of common chemicals applied in bacterial cultivation and bacteriocin purification. Dynamic of bacterial growth, acidification, and cumulation of produced bacteriocins and some aspects of the bacteriocins mode of action were evaluated. Based on obtained results, isolation and application of expressed antimicrobials can be realistic scenario for treatment of some staphylococcal associated infections. Appropriate biotechnological approaches need to be developed for cost effective production, isolation, and purification of expressed antimicrobials by studied Bacillus strains.