Discharges of Combined Sewer Overflows (CSOs) into periurban rivers present risks for the concerned aquatic ecosystems. In this work, a specific ecotoxicological risk assessment methodology has been developed as management tool to municipalities equipped with CSOs. This methodology comprises a detailed description of the spatio-temporal system involved, the choice of ecological targets to be preserved, and carrying out bioassays adapted to each compartment of the river receiving CSOs. Once formulated, this methodology was applied to a river flowing through the outskirts of the city of Lyon in France. The results obtained for the scenario studied showed a moderate risk for organisms of the water column and a major risk for organisms of the benthic and hyporheic zones of the river. The methodology enabled identifying the critical points of the spatio-temporal systems studied, and then making proposals for improving the management of CSOs.