The frequent bioaccumulation of pharmaceuticals in the aquatic ecosystem has raised a concern about their possible ecotoxicological consequences. DNA damage, haematological changes and activities of oxidative stress enzymes in Nile tilapia, Oreochromis niloticus in response to diclofenac (DCF) exposure were investigated for up to 60 days at the concentrations of 0.17, 0.34 and 0.68mgL−1 in the fish liver. Evaluation of genotoxic effects of the drug in the liver, using single-cell gel electrophoresis, showed DNA damage on exposure at the concentrations of 0.34 and 0.68mgL−1 after day 30. Compared with the control, there was a reduction in haemoglobin and red blood cell counts with a significant increase (p<0.05) in white blood cell counts, mean corpuscular volume and mean corpuscular haemoglobin level after day 30 at 0.34 and 0.68mgL−1. The levels of pack cell volume, red cell distribution width and mean corpuscular haemoglobin concentration were not significant (p>0.05) between the exposed group and the control. The indices of hepatic oxidative stress biomarkers, including lipid peroxidation and carbonyl protein, showed elevated level, depicting a positive correlation with both time and concentration. More so, activity of catalase was inhibited while reduced glutathione level decreased in the liver tissue. There was increase in the activities of superoxide dismutase, glutathione peroxidase and glutathione-S-transferase after 30 days at 0.34mgL−1. Further, activity of Na+–K+-ATPase in the tissue was significantly inhibited (p<0.05) at the end of 60 days. Prolonged exposure to diclofenac at sub-lethal concentration can cause both DNA and oxidative damages in O. niloticus, suggesting the use of oxidative stress biomarkers as early warning signals in environmental monitoring of residual pharmaceutical and assessment.