To characterize the spatio-temporal variation of sediment ecotoxicity in a retention/detention basin, a monitoring program using the Heterocypris incongruens bioassay was carried out for 72 months (5 years) on a field basin close to Lyon in France. Results showed that the variation of ecotoxicity is relatively small from one location of the basin to another, apart from sediment sampling collected in an open-air chamber located in basin supposed to collect gross pollutants and hydrocarbons. Regarding the temporal variation of ecotoxicity, the bioassays also showed a slight variation between 6 and 72 months. On the contrary, they highlighted the high ecotoxicity of the “fresh” sediments collected during rain events using sediment traps. Additional investigations are needed to understand the period of inflexion of ecotoxicity, occurring between 24 h and 6 months. These results can be used by practitioners of urban facilities and networks to improve maintenance strategies of retention/detention basins.