<p>Small to medium-sized tropical rivers in India face severe anthropogenic stress, yet remain understudied in terms of aquatic biodiversity and habitat condition. The Kana Damodar River, a tropical river in eastern India, exemplifies this problem, being increasingly impacted by sewage, industrial effluents, and seasonal water scarcity that threaten its aquatic biodiversity. This study aimed to assess the spatio-seasonal variations in fish assemblages in the Kana Damodar River and explore how hydro-environmental variables influence species composition and diversity. Monthly fish and water samples were collected from four sampling stations (S1, S2, S3, and S4) along the longitudinal fluvial gradient of the river over a two-year period (July 2020 to June 2022). Fish assemblage composition, richness, and diversity indices were analysed, and multivariate approaches (NMDS, ANOSIM, RDA) were applied to link fish communities with environmental variables. A total of 36 fish species from 18 families were identified, with fish assemblages dominated by the order Cypriniformes and the family Cyprinidae. The presence of both exotic and threatened native species in the Kana Damodar River raises significant conservation concerns. Notable variations in species richness and abundance were observed across sites, with S4 exhibiting the highest species richness and S3 the lowest. Seasonally, fish diversity and abundance declined markedly during the pre-monsoon period, when reduced river discharge led to habitat contraction and deterioration of water quality. This low-flow phase functioned as a seasonal ecological bottleneck, intensifying anthropogenic stressors and selectively favouring pollution-tolerant species, thereby compromising fish assemblages across most sampling sites. Diversity indices revealed significant spatial variations (<i>p</i> &lt; 0.05). Non-metric multidimensional scaling (NMDS) indicated distinct fish assemblages at different sites and seasons, with significant dissimilarities confirmed by analysis of similarity (ANOSIM) (<i>p</i> &lt; 0.05). Redundancy Analysis (RDA) emphasized the importance of dissolved oxygen (DO), biochemical oxygen demand (BOD), turbidity (TUR), and water temperature (WT) in shaping fish assemblages in the studied river. This study indicates that degraded water quality and seasonal low-flow conditions influence the fish assemblage structure in the Kana Damodar River. The findings of this study emphasize the urgent need for conservation efforts, including habitat restoration and pollution control, to protect this ecologically fragile river system from further degradation.</p>