The Mahanadi River holds significant ecological importance because of its large basin area of 141,600 sq. km. This basin has high potential for water resources, including rivers, lakes, reservoirs, and salt lagoons, which are often inhabited by diverse fish fauna. This study aims to investigate the diversity and distribution patterns of freshwater fish, the factors affecting fish diversity, their conservation status, and the threats to fish fauna. In total, 176 species were confirmed from various sources. The Michaelis–Menten equation can be used to predict approximately 209 species that are expected to inhabit the Mahanadi River Basin. These species are grouped into 119 genera across 59 families. The fish fauna includes one critically endangered species, five endangered species, and seven vulnerable species. The fish species exhibited a hump-shaped distribution pattern along both latitudinal and longitudinal gradients, with relatively larger areas in the mid-latitudes and mid-longitudes driving this distribution. Major threats to the fish fauna of the Mahanadi River Basin include flow modifications due to river regulation, pollution, indiscriminate fishing, and the unavailability of sufficient natural food. Given this, a substantial conservation strategy for the fish fauna of the Mahanadi River basin is warranted. Fishery scientists suggest several conservation measures, including the establishment of hatcheries for threatened species, improvements in recruitment processes, the regulation of mesh sizes in fishing nets, fish ranching, and raising awareness among fishing communities.