Chemical contamination of wastewater is a problem of great environmental concern, as it poses a hazard to both the ecosystem and to human health. In this study, we have performed a bioanalytical evaluation of the presence and removal efficiency for bioactive chemicals in wastewater treatment plants (WWTPs), using in vitro assays for toxicity endpoints of high relevance for human health. Water samples were collected at the inlet and outlet of five Swedish WWTPs, all adopting a treatment technology including pretreatment, primary treatment (sedimenation), seconday treatment (biological processes), post-sedimentation, and sludge handling. The water samples were analyzed for cytotoxicity, estrogenicity, androgenicity, aryl hydrocarbon receptor (AhR) activity, oxidative stress response (Nrf2) and the ability to activate NFĸB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling. We observed clear androgenic and estrogenic activities in all inlet samples. Androgenic and estrogenic activities were also observed in all outlet samples, but the activities were lower than the respective inlet sample. AhR activity was observed in all samples, with higher activities in the inlet samples compared to the outlet samples. The removal efficiency was found to be high for androgenic (>99% for two plants and 50-60% for two plants) and estrogenic (>90% for most plants) compounds, while the removal efficiency for AhR-inducing compounds was 50-60% for most plants and 16% for one plant.