In the present work, the conducted research concerned the determination of the toxicity and oxidative stress generation of the antidepressant fluoxetine (FLU), its metabolite nor-fluoxetine (Nor-FLU), the antibiotic nalidixic acid (NA), caffeic acid (CA) and their mixtures in three different environments: microbial medium (MM), raw wastewaters (RW) and treated wastewaters (TW). We evaluated the following parameters:E. colicell viability, toxicity and protein damage,sodApromoter induction and ROS generation. It was found that FLU, Nor-FLU, NA, CA and their mixtures are toxic and they have the potency to generate oxidative stress inE. colistrains. We also detected that the wastewater, in comparison to the microbial medium, had an influence on the toxic activity and oxidative stress synthesis of the tested chemicals and their mixtures. Regardless of the environment under study, the strongest toxic activity and oxidative stress generation were detected after bacterial incubation with NA at a concentration of 1 mg/dm3and the mixture of FLU (1 mg/dm3) with Nor-FLU (0.1 mg/dm3) and with NA (0.1 mg/dm3). The ROS synthesis andsodApromoter induction suggest that, in the case of the examined compounds and their mixtures, oxidative stress is the mechanism of toxicity. The analysis of the types of interactions among the substances constituting the mixtures in the wastewater revealed synergism, potentiation and antagonism.