Probiotic lactic acid bacteria are live microorganisms that provide health benefits when administered in adequate amounts and may exhibit antiproliferative effects on various cancer cell lines, including colon cancer. This study investigates the cytotoxic effects of three Lactobacillus strains - Limosilactobacillus (L.) reuteri VA 102, Ligilactobacillus (L.) animalis VA 105, and Limosilactobacillus (L.) reuteri KCTC 3594 (ATCC 23272) - on mouse colon carcinoma cells (CT-26). Live cells, heat-killed cells, and cell-free supernatant (CFS) of Lactobacillus sp. were prepared and used to treat CT-26 cells at different concentrations. The cytotoxic effect was assessed using the MTT assay. The results indicated that the CFS of all strains significantly reduced the viability of CT-26 cells in a dose-dependent manner, with the VA 102 strain showing the most pronounced effect. Heat-killed cells of L. reuteri VA 102 and L. reuteri KCTC 3594 (ATCC 23272) also reduced cell viability. These findings suggest the potential anticancer properties of these Lactobacillus strains and indicate that CFS and heat-killed cells may offer a safer and more effective alternative to live bacteria for therapeutic applications. Our study contributes to the understanding of the potential of Lactobacillus strains, particularly L. reuteri VA 102, L. reuteri KCTC 3594 (ATCC 23272), and L. animalis VA 105, as possible candidates for cancer treatment and control.