The genotoxicity of water and sediment samples from stormwater treatment systems and water from urban highway runoff was tested in the Salmonella/microsome assays with Salmonella typhimurium, micronucleus assay (Trad-MN) with plants and with human-derived liver cells (HepG2), or comet assay with HepG2. Cytotoxicity of water samples was studied using either reactive oxygen species (ROS) generation, cell proliferation or dye exclusion assay in HepG2. Concentrations of several contaminants in the tested samples were also measured. Results suggested that urban highway runoff exposed to severe vehicle traffic emissions caused genotoxic effects in comet assay and in Trad-MN assays. Sediments induced either mutagenic effects in strain YG1024 or genotoxic effects in Trad-MN assay. These effects could be due to the presence of nitro-polycyclic aromatic hydrocarbons (NPAHs) which possess carcinogenic and mutagenic properties. Influent and effluents of stormwater treatment systems did not induce genotoxic activity or effects on HepG2 cell viability; however, the influents were able to induce ROS generation and cell proliferation in HepG2 cells. As the methods require a sterile filtration of the water samples, this could have also removed particulate-associated polycyclic aromatic hydrocarbons (PAHs) and resulted in a less pronounced induction of genotoxicity, as would be expected by PAH contamination.