Indirect potable reuse of wastewater is a practice that is gaining attention, aiming to increase freshwater supplies to meet water scarcity. However, reusing effluent wastewater for drinking water production comes with a paired risk of adverse health effects, due to the potential presence of pathogenic microorganisms and hazardous micropollutants. Disinfection is an established method to reduce microbial hazards in drinking water, but it has been associated with formation of disinfection by-products (DBPs). In this study, we performed an effect-based assessment of chemical hazards in a system wherein a full-scale trial of disinfection by chlorination, of the treated wastewater was performed prior discharge to the reciepient river. The presence of bioactive pollutants was assessed along the entire treatment system, starting from incoming wastewater to finished drinking water at seven sites in and around the Llobregat River in Barcelona, Spain. Samples were collected in two campaigns, with and without applied chlorination treatment (13 mg Cl2/L) to the effluent wastewater. The water samples were analysed for cell viability, oxidative stress response (Nrf2 activity), estrogenicity, androgenicity, aryl hydrocarbon receptor (AhR) activity and activation of NFĸB (nuclear factor kappa-light-chain-enhancer of activated B cells) signaling using stably transfected mammalian cell lines. Nrf2 activity, estrogen receptor activation and AhR activation was detected in all investigated samples. Overall, removal efficiencies were high in both wastewater treatment and drinking water treatment samples for most of the studied endpoints. No increase in oxidative stress (Nrf2 activity) could be attributed to the additional chlorination treatment of the effluent wastewater. However, we found an increase in AhR activity and a reduction of ER agonistic activity after chlorination treatment of effluent wastewater. The bioactivity detected in finished drinking water was considerably lower compared to what was found in effluent wastewater. We could thus conclude that indirect reuse of treated wastewater for drinking water production can be possible without compromising drinking water quality. This study contributed important knowledge in efforts to increase the reuse of treated wastewater as a source for drinking water production.