Background: Anti-proliferative effects of probiotics are considerable in the treatment of various cancers, including colon cancer. In the present study, two new Lactobacillus strains as probiotics were isolated from stool samples at a clinical lab. Objectives: The aim of this study was to evaluate the effects of the cell-free lyophilized filtrate of two new strains of Lactobacillus, isolated on viability on Caco-2 cells. Methods: Two new strains of Lactobacillus were isolated from 1 gr of each infant stool specimens from a total of fifty volunteers, according to the principles of a scientific questionnaire. The anti-proliferative effects of the strains were investigated using the MTT assay with Caco-2 cell lines. Results: Out of 50 samples, seven isolates were lactic acid bacteria, two strains of which were probiotics related to L. fermentum (E) and L. rhamnosus (G). The results showed that the two Lactobacillus strains had good anti-proliferative effects against the cancer cell lines tested. These strains were resistant to low pH and 0.3% bile salt. Cytotoxicity assay revealed that the most effective concentration of strains E (~55% to ~72%) and G (~60% to ~80%) on Caco-2 cells was 10000 µg/mL after 24 to72 hours. Conclusions: Cytotoxicity effect of the cell-free lyophilized filtrate of bacteria on Caco-2 cells in a dose- and time-dependent manner suggested that these strains might be used in colon cancer therapy.