The present study carried out to clarify the impact of heavy metals (Fe, Zn, Cu, Mn and Cd) pollution of Rosetta Branch on the antioxidant defence system activities and lipid peroxidation indicator MDA levels in O. niloticus tissues (liver and white muscles) collected from three stations from Rosetta Branch of River Nile in summer 2014 and winter 2015. Rosetta Branch of River Nile exposed to high input of agricultural drainage water, sewage and industrial waste water which influence the living organisms especially fish. In the present study, results revealed that high concentrations of heavy metals (Fe, Zn, Cu, Mn and Cd) were detected in water and fish samples, especially in winter. In muscles of O. niloticus, the accumulation patterns of heavy metals were in the following order: Fe > Zn > Mn > Cu and Cd. The bioaccumulation factor (BAF) in winter was higher than summer. Antioxidant enzymes (SOD, CAT, GPx, GST and GR) activities and the indicator of lipid peroxidation MDA levels in liver and white muscles of O. niloticus were found to be significantly increased compared to the reference values, especially in winter. Moreover, the antioxidant enzymes activities and MDA levels were higher in liver than white muscles. These remarkable alterations in the activity of the selected enzymes in the liver and white muscles of the O. niloticus go in parallel with the elevation in the levels of heavy metals detected in the water of Rosetta branch, as a result of pollution stress in these areas. Thus we conclude that, the altered activities of SOD, CAT, GPx, GST and GR) and MDA levels could be useful biomarkers of water pollution.