Pollution of aquatic ecosystems is associated with the discharge of mainly industrial and urban effluents, which may cause damage to public health. This study aims to evaluate the cytotoxic, genotoxic, and mutagenic potential of surface water samples under the influence of different anthropogenic effluents in a human-derived liver cell line (HepG2). Samples were collected in Esteio and Sapucaia streams (Rio Grande do Sul; Brazil), which flow into the Sinos River, a source of water supply for more than one million people. Physicochemical and microbiological analyses were performed as well as an analysis of inorganic elements using the PIXE technique (Particle-Induced X-Ray Emission). The presence of pharmaceutical compounds and caffeine was evaluated by gas chromatography coupled to mass spectrometry. The cytotoxicity, genotoxicity, and mutagenicity of the samples were evaluated in HepG2 cells by cell viability assays, alkaline Comet Assay and Cytokinesis-block micronucleus (CBMN) assay. We verified alterations in the physicochemical and microbiological parameters and detected caffeine, diethyltoluamide, and different inorganic elements that corresponded to elements from domestic and industrial effluents and agricultural runoff. Although the samples in the concentration used were not cytotoxic, water samples from all sites induced DNA damage. However, it is difficult to attribute these damages to a specific substance since the factors are a complex mixture of different compounds. Despite this, it is observed that both urban and industrial contributions had a similar effect in the cells evaluated. Such results demonstrate the need to perform biomonitoring of surface waters under anthropogenic influence, especially those that flow into rivers that are a source of public supply water. We also highlight the need for research into emerging pollutants in these aquatic environments.