Conventional activated sludge treatment of wastewater does not completely remove micropollutants. Here, extending anaerobic conditions may enhance biodegradation. To explore this, we combined iron-reducing or substrate-limiting and aerobic pilot-scale reactors directly at a wastewater treatment plant. To assess the removal of endocrine disrupting chemicals (EDCs) as group of micropollutants that adversely affects wildlife, we applied a bioanalytical approach. We used in vitro bioassays covering seven receptor-mediated mechanisms of action, including (anti)androgenicity, (anti)estrogenicity, retinoid-like, and dioxin-like activity. Untreated wastewater induced antiandrogenic, estrogenic, antiestrogenic, and retinoid-like activity. Full-scale as well as reactor-scale activated sludge treatment effectively removes the observed effects. Nevertheless, high antiandrogenic and minor dioxin-like and estrogenic effects persisted in the treated effluent that may still be environmentally relevant. The anaerobic post-treatment under substrate-limiting conditions resulted in an additional removal of endocrine activities by 17-40%. The anaerobic pre-treatment under iron-reducing conditions significantly enhanced the removal of the residual effects by 40-75%. In conclusion, this study demonstrates that a further optimization of biological wastewater treatment is possible. Here, implementing iron-reducing anaerobic conditions preceding aerobic treatment appears promising to improve the removal of receptor-mediated toxicity.