Oxytetracycline (OTC), an antibacterial agent, is extensively used in aquaculture practices all over the world, but also in human and veterinary medicines. Because of its intensive use, low rates of absorption by treated animals, inadequate disposal, and low efficiency of removal in wastewater treatment plants, the potential harmful effects on aquatic organisms are of great concern. This work aimed to assess the effects of this antibiotic in rainbow trout, following both acute and chronic exposures. Catalase (CAT), total glutathione peroxidase (GPx), glutathione reductase (GRed) activities and lipid peroxidation (TBARS levels) were quantified as oxidative stress biomarkers, in gills and liver. Genotoxic endpoints, reflecting different types of genetic damage in blood cells, were also determined, by analysis of genetic damage (determination of the genetic damage index, GDI, measured by comet assay) and erythrocytic nuclear abnormalities (ENAs). The obtained results showed a mild pattern of antioxidant response, with modifications in CAT and GPx activities in gills, and lipid peroxidation in liver. These results suggest that despite the occurrence of oxidative effects, a full scenario of oxidative stress is not likely. However, exposure to OTC resulted in the establishment of genotoxic alterations with the induction of DNA strand breaks in blood cells (increase of GDI), and of chromosome breakage and/or segregational abnormalities (increase of ENAs). Considering that the oxidative response was not totally devisable, other mechanisms may be involved in the genotoxic effects reported.