Background: Leukemia remains a global health challenge, requiring the exploration of alternative therapies with reduced side effects. Probiotics, particularly Lactobacillus species, have gained attention because of their potential anticancer properties. This study investigated the anticancer and cytotoxic effects of postbiotic mediators (PMs) derived from Lactobacillus rhamnosus GG (LGG) and Lactobacillus reuteri (LR) on acute lymphoblastic leukemia (ALL) cells and peripheral blood mononuclear cells (PBMCs). Materials and Methods: The PMs were prepared by culturing LGG and LR strains and isolating the supernatant. The MTT assay assessed cell viability on ALL Jurkat cells and PBMCs, and apoptosis analysis was conducted using flow cytometry. Quantitative real-time PCR was also performed to analyze BAX, BCL-2, BCLX, FAS, and p27 gene expression levels. Results: The results showed that PMs derived from LGG and LR significantly reduced cell viability in Jurkat cells (P<0. 05) but not PBMCs (P>0. 05). Apoptosis analysis revealed an increase in apoptotic cells after PMs treatment. Nevertheless, gene expression analysis revealed no statistically significant difference between the treated and untreated groups in BAX, BCL-2, BCLX, FAS, and p27 gene expression levels (P>0. 05). Conclusion: Findings suggest that specific PMs derived from LGG and LR possess anticancer properties against ALL cells. This research highlighted the promise of PMs as a cutting-edge and less toxic adjuvant therapeutic strategy in cancer treatment.