A set of CALUX in vitro bioassays was applied for long-term toxicity monitoring at an advanced wastewater treatment plant comprising ozonation and granular activated carbon filtration for the abatement of contaminants of emerging concern (CEC). During the 13-month monitoring, eight reporter gene assays targeting different modes of action along the cellular toxicity pathway were accessed to evaluate the suitability and robustness of the technologies. Two approaches were followed: on the one hand, signal reduction during advanced treatment was monitored; on the other hand, results were compared to currently available effect-based trigger values (EBTs). A decrease of the corresponding biological equivalent concentrations after the multibarrier system could be observed for all modes of action; while the estrogenic activity decreased below the EBT already during ozonation, the potencies of oxidative stress-like and toxic PAH-like compounds still exceeded the discussed EBT after advanced treatment. Overall, the long-term monitoring confirmed the positive effect of the multibarrier system, commonly evaluated only by CEC abatement based on chemical analysis. It could be demonstrated that advanced WWTPs designed for CEC abatement are suitable to significantly decrease toxicity responses not only in the frame of pilot studies but under real-world conditions as well.