The Sinos River is one of the most polluted rivers in Brazil. The purpose of this work was to monitor the presence of some pesticides and hydrocarbons as well as the genotoxic and cytotoxic potential on HEp-2 cells from water samples collected at seven sites in the Sinos River Basin (SRB), southern Brazil. Nine samples were taken from the three main rivers in the SRB and used as a solution to dilute the HEp-2 cell culture medium after microfiltration. Twenty-four pesticides and 19 hydrocarbons were measured. Cytotoxicity was assessed by methyl thiazolyl tetrazolium (MTT) and neutral red (NR) assays, in which cells were exposed to different concentrations of the water samples for 24 h. Genotoxicity of the microfiltrated raw water samples was assessed by comet assay after 6 and 24 h of exposure. Among the chemicals analyzed, only the 2,4-D, dichloromethane, tetrachloroethene, chloroform, bromodichloromethane, styrene, and toluene were detected, but they were all lower than the limit established by Brazilian regulations. Twenty samples from a total of 60 had a cytotoxic effect in the MTT assay and 30 in the NR assay. The comet assay indicated the presence of genotoxic substances in the water at the seven locations monitored. Temporal and spatial variation was observed in the cytotoxicity and genotoxicity assays. Results indicated that the water in all stretches of the SRB is contaminated and it can cause harmful effects to humans and to the aquatic biota. This HEp-2 cell-line approach can be an additional tool for environmental monitoring.