Cancer constitutes an important part of the diseases that are caused all over the world. Due to the side effects of commonly used treatment options such as chemotherapy and radiotherapy, there has been a tendency to seek natural anticancer compounds. In this study, the cytotoxic effects of L. plantarum AB625, L. plantarum MK55, and S. boulardii T8-3C probiotic strains on HCT-116, HeLa, and MDA-MB-231 cell lines were determined. The effect of the microorganisms' cell contents was also evaluated in 3D cancer cell lines. To evaluate the effect of microorganisms on metastasis, we applied to wound healing and invasion assays. At the same time, the cytotoxic effect of microorganisms on cancer cell lines was determined at the gene level using Real-Time qPCR. In addition, the effect of microorganisms on the expression of CASP3 protein in HeLa cells was also evaluated. At the last stage of the study, molecular docking and molecular dynamics simulation studies were carried out with the microorganism metabolites detected by QTOF-LC/MS. The results of the study showed that the most effective strain on HeLa and MDA-MB231 cells was L. plantarum AB6-25 with a 91% and 80% inhibition rate, respectively, while L. plantarum MK55 had the highest cytotoxic effect on HCT-116 cells with a 69% inhibition rate. On the other hand, S. boulardii T8-3C had a weaker anticarcinogenic effect on cancer cell lines compared to the other two bacteria. According to the migration and invasion assays results, HCT-116 and MDA-MB-231 cells were the most effective bacteria in preventing L. plantarum MK55, and S. boulardii T8-3C metastasis in HeLa cells. As a result of gene expression analysis, microorganisms showed the greatest effect on apoptotic genes in HeLa cells. In HCT-116 and MDA-MB-231 cells, the expression level of apoptotic genes changed less. In the western blot analysis, L. plantarum AB6-25, L. plantarum MK55, and S. boulardii T8-3C cell contents expressed CASP3 at a higher level in the 2D HeLa cells compared to the 3D cells. The BAX protein and istamycin C1 ligand, which resulted in the binding energy of-8.23 kcal/mol, were determined as the molecules with the best anti-cancer effect. The results of in vitro studies have been supported by genetic expression analysis and molecular docking studies, and have shown the potential of microorganisms' cell contents to be an anticancer agent. (c) 2022 Elsevier B.V. All rights reserved.