Abstract The genotoxicity and cytotoxicity of water in small urban basins was evaluated by the Salmonella/microsome assay and micronucleus test in V79 cells. The results showed that the cytotoxic effect was the most significant response in areas with medium to heavy urban occupation for both assays evaluated. Water samples from these areas include different concentrations of chloroform, bromodichloromethane, toluene, ethylbenzene, m, p-xylene and 1,4-dichlorobenzene. As to genotoxic damage, the presence of mainly direct-acting frameshift mutagens was detected in areas with less urban concentration and showed genotoxic activity in V79 cells in more heavily urbanized areas. Water organic extracts, evaluated using a microsuspension procedure, showed frameshift mutagenic activity in the presence of hepatic metabolization that increased as the population density grow. Chronic toxicity studies of sediment samples with the microcrustacean Daphnia magna showed that, while survival was not highly affected, reproductive inhibition was found in 92% of the observations. A retrospective diagnosis of water quality using traditional physicochemical parameters that defined the differential contribution of urban wastes at the three sites was associated with the biological assays. It became clear that the biological assays were of significant benefit in the diagnosis of risks of contamination of hydrographic basins by pollutants from urban non-point sources.