The main purpose of this study is to evaluate the impact of heavy metals (Pb, Zn, Cu, Fe, and Cd) aquatic pollution of the EL-Mahmoudeyia canal on the antioxidant enzymatic activities, GSH content, and lipid peroxidation levels (MDA) in Oreochromis. niloticus muscle tissues collected from two areas EL-Mahmoudeyia canal as Polluted area and  Rosetta branch of river Nile as reference area in summer 2018 and winter 2019 as well as DNA damage was assessed in fish gills(erythrocytes) samples by applying comet assay. EL-Mahmoudeyia canal exposed to excessive industrial effluents which impact the living organisms especially fish. The herein results showed that higher concentrations of heavy metals (Pb, Zn, Cu, Fe, and Cd) were detected in water and fish samples collected from the polluted area in comparison with the reference area, especially in winter.  The accumulation patterns of heavy metals in the muscles of O. niloticus, were in the following order: Fe > Zn >Pb> Cu and Cd.The antioxidant enzymatic activities of (SOD, CAT, GPx, and GST) and the lipid peroxidation biomarker MDA levels in muscles of O. niloticus collected from the polluted area were found to be significantly increased compared to that of the reference area. Meanwhile, there was a significant decrease in the GSH content level in the muscles of O. niloticus collected from the polluted area compared to that of the reference area. A significant elevation in DNA damage frequencies was observed in fish collected from the polluted areas compared with those from the reference area. These noticeable alterations in the selected antioxidant enzymatic activities in muscles of the O. niloticus go in parallel with the remarkable elevation in the levels of the detected heavy metals in water from EL Mahmoudeyia canal, as a result of pollution in these areas. This study explored the utility of the DNA damage, the altered antioxidant enzymatic activities, GSH content, and MDA level as biomarkers of aquatic pollution.