Potential probioticEnterococcus faecalisKUMS-T48, isolated from a kind of Iranian traditional dairy product (Tarkhineh), was assessed for its anti-pathogenic, anti-inflammatory and anti-proliferative properties against HT-29 and AGS cancer cell lines. This strain showed strong effects onBacillus subtilisandListeria monocytogenesand moderate effect onYersinia enterocolitica, while indicated weak effect onKlebsiella pneumoniaeandEscherichia coli. Also, neutralizing the cell-free supernatant and treating it with catalase and proteinase K enzymes reduced the antibacterial effects. Similar to Taxol, the cell-free supernatant ofE. faecalisKUMS-T48 inhibited the in vitro proliferation of both cancer cells in a dose-dependent manner, but unlike Taxol, they had no activity against normal cell line (FHs-74). Pronase-treatment of the CFS ofE. faecalisKUMS-T48 abrogated its anti-proliferative capacity, thereby showing the proteinaceous nature of the cell-free supernatant. Further, induction of apoptosis-based cytotoxic mechanism byE. faecalisKUMS-T48 cell-free supernatant is related to anti-apoptotic genes ErbB-2 and ErbB-3, which is different from Taxol’s apoptosis induction (intrinsic mitochondria apoptosis pathway). Also, as evidenced by a decline in interleukin 1β inflammation-promoting gene expression and a rise in the anti-inflammatory interleukin-10 gene expression in the HT-29 cell line, probioticE. faecalisKUMS-T48 cell-free supernatant demonstrated a significant anti-inflammatory impact.