The River Nile is the primary source of freshwater for drinking, irrigation, and industrial purposes in Egypt. Thus, the water quality in this river concerns the health of local inhabitants. The present study reveals seasonal variations of various physicochemical and heavy metals parameters and microbial load of water at 15 sites from Qena to Sohag cities, Egypt. The water is fresh with TDS <= 270 and 410 mg L-1 in summer and winter, respectively. Fe, Mn, Cd, Cr, Cu, Ni, and Zn concentrations were within drinking water specification in both seasons except Cr and Cd in summer. Viable numbers of total coliform, fecal coliform, and fecal streptococci were recorded in both seasons with fecal streptococci's disappearing in winter. The concentrations of salts and ions in winter were higher than summer due to decreased water quantity and flow rate in this season. On the other hand, heavy metals and bacteria were higher in summer owing to the rain and weathering of upstream rocks and increasing of human activities during the summer. The calculated water quality index (WQI) depicted that the chemical quality of water was poor for drinking and treatment, especially biological treatment, which is required before the water is supplied for drinking. Human health risk assessment factors such as probable daily intake, hazard quotient, and carcinogenic risk indicated high risks of Cr, Cd, and Ni for adults and children in both seasons. The non-carcinogenic and carcinogenic risks are mainly posed by Cr. The WQI values for the other water uses indicated the marginal quality for aquatic life, fair for irrigation, and fair in summer to good in winter for livestock consumption. The irrigation water quality parameters indicated that the water could be used to irrigate all soils and crops except the hazard of biological contamination. The water-rock interaction controls water chemistry besides the contribution of human activities. The agricultural, industrial, and municipal wastewaters were the main contributors to water pollution and should be treated before discharge into the Nile River. Source and drinking water should be monitored continuously to prevent related human waterborne diseases.