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GSH (glutathione) is crucial for the removal and detoxification of carcinogens in healthy cells, while in cancer cells, GSH is associated with cancer expansion and increased resistance to drugs. O₂^•− acts as a secondary messenger and plays a major role in the cell signalling pathways of normal and cancer cells. Herein, the levels of O₂^•− and GSH were measured in MRC-5 and HCT-116 cells after incubation with BAL (<i>Bifidobacterium animalis</i> spp. <i>lactis</i>) and BAL/EALS (ethyl acetate extract of <i>Laetiporus sulphureus</i>) in co-culture systems, and for the first time, sensitivity was compared between these cell lines. The O₂^•− and GSH parameters were measured spectrophotometrically after 12 and 24 h. The levels of the O₂^•− were slightly increased in the MRC-5 cells after the effect of BAL and BAL/EALS (10 µg/mL), while the highest concentration of O₂^•− was recorded in treatment with BAL/EALS (50 µg/mL). On the other hand, the GSH values were elevated already after 12 h of incubation, and then further increased after 24 h in the MRC-5 cells. In the HCT-116 cells, the concentration of O₂^•− was not enhanced at 12 and 24 h of incubation compared to that of the control. The GSH level also remained relatively low. We observed a positive dose-dependent effect on the GSH levels in the MRC-5 and a negative dose-dependent effect in the HCT-116 cells. Generally, high GSH levels in the MRC-5 after 12 and 24 h indicate a strong reaction to oxidative stress and more sensitivity compared with the HCT-116 cells, where GSH stayed at a low concentration.