Yamuna River water in Agra city of India is heavily contaminated with toxic pollutants, including heavy metals that cause severe damage to ecological and social aspects of life. At present, the direct use of river water for drinking purposes cause severe hazards due to anthropogenic activities and environmental pollution. The quality assessments in terms of physical, chemical, and biological attributes of water samples are essential to understand their impact on humans. In the present study, Yamuna River water samples were collected from three different sites in Agra city during the monsoon, summer, and winter seasons. Various physico-chemical parameters were estimated, and the concentration of heavy metals was measured. In the case of physico-chemical parameter characterization, the values found were mostly above the permissible limits. The results suggest that the Yamuna River water samples contain high levels of cadmium, chromium, lead, and nickel above the desirable levels in most cases. The water samples were subjected to metagenomic analysis using the Illumina platform, which revealed that Proteobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria, and Planctobacteria were the most abundant phyla with a relative abundance of 61%, 9.34%, 5.23%, 4.64%, and 4.3%, respectively. The Comamonadaceae was the most abundant family, which consists of the genera involved in hydrogen oxidation, iron reduction, degraders of polycyclic aromatic hydrocarbons, and fermentation. Functional prediction showed the presence of genes responsible for different metabolic pathways. The study concludes the status of water contamination at three different sites in Agra and suggests the futuristic use of this research in the eld of bioremediation.