Water pollution is one of the most principal environmental and public health problems in the River Nile. In the present study, aims were directed to evaluate the biochemical, histopathological, and genetic effects of El- Rahawy Drain pollutants on the Nile tilapia (Oreochromis niloticus). Hence, during the summer of 2018 and winter 2019, fish samples were collected from two locations (upstream and downstream) of El-Rahawy drain in the River Nile at the Rosetta Branch. Biochemical studies (Total Protein, Albumin, Globulin, A/G, total lipids, Cholesterol, AST, and ALT) were carried out for the specimens and important results were revealed. Compared with the fish caught from the upstream, a deterioration was detected in the health status of the downstream- fish; displaying more extreme histopathological and molecular variations. Using SSR (simple sequence repeats) to detect the molecular effects of water pollutants on O. niloticus, molecular markers indicated genetic differences in fish samples from the same sex in the different studied locations. Consequently, to prevent disease outbreaks and aquatic ecosystem disturbances, the Rosetta Branch is recommended to have an intensive water-quality monitoring program, and wastewater treatment as well.