OBJECTIVE Bacillus probiotics have been recently considered in biotechnological researches, and food additives. The present study was aimed to investigate the effects of Bacillus subtilis probiotics (PY79 and ATCC 6633) and their metabolites on Salmonella Typhimurium in Caco-2 cells. METHODS Cytotoxicity of B. subtilis ATCC 6633 crude supernatant (CS) was evaluated by 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. S. Typhimurium invasion assay was performed in the presence of the probiotics. Cell viability, apoptosis, and necrosis were evaluated in presence of S. Typhimurium, B. subtilis strains, and CS (4%, 8%) using flow cytometry. RESULTS Results showed a significant reduction in the invasive ability of S. Typhimurium to Caco-2 cells by employing B. subtilis probiotics, and CS (p < 0.05). The less invasion was indicated in B. subtilis PY79 and Salmonella co-cultural group. Furthermore, the cell survival rates, and apoptosis/necrosis were respectively increased and decreased in co-culture groups (p < 0.05). CONCLUSIONS Hence, it seems that B. subtilis strains could be suggested as beneficial candidates to overcome the invasion and cytotoxicity of Salmonella on the intestinal cells. However, additional in vivo models are suggested to validate our results.