In this study, we evaluated the effects of prolonged exposure to two different nano-TiO2 crystal phases under different illumination conditions. Fish (Piaractus mesopotamicus) were exposed for 21 days to 100 mg/L of nano-TiO2 anatase and a mixture of anatase:rutile (80%:20%) under visible light and UV light (UVA and B, 22.47 J/cm2/h). The following oxidative stress biomarkers were mon- itored: concentrations of lipid hydroperoxide (LPO), carbonylated proteins (PCO), and specific activ- ities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST). Other biomarkers as well as specific activities of acid phosphatase (AP), Na+, K(+)-ATPase and metalloth- ionein levels (MT) were also evaluated. Moreover, micronucleus and comet assays were performed to assess genotoxicity. Our results showed low toxicity of nano-TiO2 to fish and lack of titanium accumulation in muscle tissue. However, it was observed the occurrence of sublethal effects that were influenced by nano-TiO2 crystal phase and illumination condition. Pure anatase caused more oxidative damage without co-exposure to UV, while the mixture anatase:rutile caused more sub- lethal effects when exposure occurred under UV. These findings show that the specific activity of CAT, GST, PCO levels and comet assay are useful as biomarkers of prolonged exposure to nano- TiO2. Overall, our study substantiates the development and implementation of nanoecotoxicological protocols.