Very few tools are available for assessing the im- pact of combined sewer overflows (CSOs) on receiving aquat- ic environments. The main goal of the study was to assess the ecotoxicological risk of CSOs for a surface aquatic ecosystem using a coupled “substance and bioassay” approach. Wastewater samples from the city of Longueuil, Canada CSO were collected for various rainfall events during one summer season and analyzed for a large panel of substances (n = 116).Fourbioassayswerealsoconductedonrepresenta- tive organisms of surface aquatic systems (Pimephales promelas, Ceriodaphnia dubia, Daphnia magna, and Oncorhynchus mykiss). The analytical data did not reveal any ecotoxicological risk for St. Lawrence River organisms, mainly due to strong effluent dilution. However, the substance approach showed that, because of their contribution to the ecotoxicological hazard posed by the effluent, total phospho- rus (Ptot), aluminum (Al), total residual chlorine, chromium (Cr), copper (Cu), pyrene, ammonia (N–NH4+), lead (Pb), and zinc (Zn) require more targeted monitoring. While chronic ecotoxicity tests revealed a potential impact of CSO dis- charges on P. promelas and C. dubia, acute toxicity tests did not show any effect on D. magna or O. mykiss, thus underscoring the importance of chronic toxicity tests as part of efforts aimed at characterizing effluent toxicity. Ultimately, the study leads to the conclusion that the coupled “substance and bioassay” approach is a reliable and robust method for assessing the ecotoxicological risk associated with complex discharges such as CSOs.