Identifying the key determinants and their impacts on water quality and fish community structure is imperative to environmental assessment and ecosystem conservation. The main objectives of this study were to assess the factors driving water quality, fish assemblages, and ecological health in a temperate lotic ecosystem using a long-term (12 years) dataset. The results indicate that sewage treatment plants significantly negatively impact river water quality and are primary pollution sources. Temporal fluctuations showed that the summer monsoon adversely influenced TN and EC due to dilution effects while increasing the COD and TSS in the water. Annual variations and the Mann–Kendall test demonstrated increasing trends in COD, TN:TP ratio, and CHL-a but a decreasing trend of TP with the impoundment. Artificial barriers (weirs) in the river created more favorable algal growth conditions due to increased water residence time. COD and TSS levels in the river were significantly affected by soil erosion. The environmental disturbance measures index indicated that all sites were severely degraded. Fish composition analysis suggested that exotic fish species such as, Lepomis macrochirus and Micropterus salmoides, dominated the community structure and negatively impacted native species after the installation of weirs. Our findings indicate that fish guilds and the IBI model are controlled by nutrient enrichment, organic matter, algal production, and land use/land cover. Water quality governs the biological health of the river. Overall, evaluation of the river's ecological health based on the multi-metric WPI and the IBI revealed that the river has a “poor” to “very poor” ecological status. The outcomes of this study may aid policymakers in managing and restoring the river.