Bifidobacterium animalis subsp. lactis (BAL) is a well-known probiotic. Laetiporus sulphureus is an edible mushroom with a recognizable effect on human health. A single and synergistic effect of BAL and BAL/ethyl acetate extract of L. sulphureus (EALS) on the levels of (superoxide anion radical) O₂^-. and (reduced glutathione) GSH in (healthy lung fibroblast) MRC-5 and (colorectal cancer cells - CRC) HCT-116 cells have been poorly explored. It is known that increased production of O₂^-. underlies carcinogenesis. GSH is crucial for the maintenance of redox balance in cells, and it is the first defense line from O₂^-.. Our study aimed to investigate the effect of BAL, and co-treatment BAL/EALS (concentrations 10 and 50 µg/mL) on O₂^-. and GSH levels in the MRC-5 and HCT-116 cells. Incubation of the MRC-5 and HCT-116 cells with the probiotics and EALS extract was performed in a simple co-culture system. The O₂^-. and GSH levels were measured spectrophotometrically after 12 and 24 h. The levels of O₂^-. and GSH were increased in the treated MRC-5 cells, especially after the BAL/EALS (50 µg/mL) co-treatment. In the treated HCT-116 cells, O₂^-. remained at a low level and GSH concentration decreased after 12 h. After 24 h of incubation, the BAL and BAL/EALS increased the level of GSH in the treated cells when compared with control (untreated) cells. High GSH levels after 12 h indicate that the MRC-5, as healthy cells, strongly reacted to changes in oxidative stress parameters. So, these cells were more sensitive to treatments when compared with the HCT-116 cells isolated from CRC. Lower O₂^-. and GSH values in the HCT-116 cells, especially at 12 h, show oxidative stress resistance which is one of the main characteristics of CRC cells. These results can be explained by the fact that MRC-5 cells do not physiologically interact with BAL, while HCT-116 cells are epithelial cells from CRC and these cells are familiar with the presence of BAL in human colon.