Probiotics, as live microorganisms, play a crucial role in maintaining microbial balance and reducing the growth of spoilage bacteria in food products. The supernatants obtained from probiotic cultures contain bioactive compounds such as organic acids, hydrogen peroxide, and bacteriocins, which can serve as natural alternatives to chemical preservatives in the food industry. The aim of this study was to evaluate the inhibitory effect of the supernatants derived from Lactobacillus acidophilus, Pediococcus acidilactici, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus buchneri, Bacillus subtilis, Bacillus coagulans, Enterococcus faecium, and Bacillus licheniformis against foodborne spoilage and pathogenic bacteria, including Escherichia coli, Salmonella typhimurium, Proteus mirabilis, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa and Bacillus cereus. To assess antimicrobial activity, probiotic supernatants were extracted and tested against target pathogens using a 96-well microplate assay. Bacterial growth was determined by optical density measurement at 600 nm, and the percentage of growth inhibition was calculated accordingly. The findings indicated that the supernatants of Lactobacillus acidophilus, Pediococcus acidilactici and Lactobacillus rhamnosus exhibited the highest inhibitory effects compared to other strains. These results highlight the potential of probiotic-derived metabolites in controlling spoilage microorganisms and enhancing food shelf life. In conclusion, this study demonstrates the high potential of certain probiotic strains in producing bioactive compounds with antimicrobial properties, suggesting their application as natural alternatives to chemical preservatives in the food industry. Future research is recommended to investigate the stability of probiotic supernatants under different food processing and storage conditions, to elucidate the exact antimicrobial mechanisms of the secreted compounds.