Bifidobacterium animalis subsp. lactis (BAL) is a commercial probiotic. It is known for its application in regulating the human gut microbiome composition. Nowadays, scientific research reveals cytotoxic effect of the BAL on colorectal cancer (CRC) cell lines. Laetiphorus sulphureus (LS) is an edible mushroom that produces various secondary metabolites with biological activity. However, as far as we know, the effect of BAL in a co-treatment with LS was not investigated previously on CRC cells, especially on HCT-116 cell line, therefore, this was the aim of our study. We used LS ethyl acetate extract (EALS) in two selected concentrations (10 and 50 µg/mL). The HCT-116 cell viability was estimated in a simple co-culture system (modified HoxBan system) in treatment with the BAL and BAL/EALS. The effect of EALS on the viability of these cells without BAL was also tested. The cell viability was determined with trypan blue staining after 12 and 24 h. Levels of redox status parameters (H₂O₂ and NO₂^-) were determined after 12 and 24 h in HCT-116 cells with BAL and BAL/EALS in order to determine the cause of HCT-116 cell death. Our results showed the significant cytotoxicity of the BAL on the HCT-116 cells in a co-culture system, while the EALS extract further enhanced the cytotoxicity of this probiotic. Dose-dependent effects were noticed in a co-treatment consisting of BAL and EALS. Namely, the lowest viability was noticed at 50 µg/mL of EALS. Considering that EALS extract did not cause a cytotoxic effect in monocultures, it can be concluded that EALS stimulated the anticancer activity of BAL. We detected higher H₂O₂ and NO₂^- values in treatment with BAL, especially in co-treatment BAL/EALS. The death of the HCT-116 cells may be due to elevated levels of NO₂^- and H₂O₂ and possibly their products (peroxynitrites). Further studies will reveal the more detailed mechanism by which the BAL and BAL/EALS treatments cause death of the HCT-116 cells.