Copper oxide nanoparticles (CuO NPs) are used for their biocide potential however they were also shown to be highly toxic to mammalian cells. Therefore, the effects of CuO NPs should be carefully investigated to determine the most sensitive processes for CuO NP toxicity. In this study, the genotoxicity of CuO NPs was investigated in vitro, using the mouse neuroblastoma cell line Neuro-2A. Genotoxic effects related to DNA fragmentation, DNA methylation and chromosomal damage, as well as lipid peroxidation, were investigated and compared to cytotoxic effects, measured by the mitochondrial reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide into formazan. Based on mitochondrial activity, CuO NPs were found to be cytotoxic. At the highest concentration tested (400 mg l⁻¹), 63% of cell viability was found in Neuro-2A cells after 24 h of treatment to CuO NPs. CuO NPs were also found to induce DNA fragmentation, lipid peroxidation and micronucleus formation. The micronucleus assay was the most sensitive to evaluate CuO NP genotoxicity and micronucleus frequency was increased significantly at 12.5 mg l⁻¹ CuO NPs after 24h of treatment. At this concentration, no significant change of cell viability was found using the mitochondrial activity assay. These results highlight the important risk of genotoxic effects of CuO NPs and show that genotoxicity assays are a sensitive approach to evaluate the risk of CuO NP toxicity.