Degradation of fish diversity in the freshwater habitats, especially the rivers of Bangladesh, has become a major concern in recent years. Therefore, this study was conducted in the Gurukchi River of Gowainghat sub-district in the Sylhet district of Bangladesh between March 2018 and February 2019 to assess the availability and biodiversity of fish species with conservation status, and population trends, and threats. Data were collected on a monthly basis by direct catch observation and field surveys as focus group discussions and personal interviews of different types of stakeholders. A total of 55 indigenous and two exotic fish species belonging to 9 orders, 22 families, and 42 genera were identified. Cypriniformes was the dominant order, comprising 40.35% of the total documented fish species, whereas Cyprinidae was found as the richest family (33.33%). A total of 10 threatened fish species (18.18%) were found in the study area where 5 species (9.09%) were endangered and five species (9.09%) were vulnerable. Notably, four globally threatened fish species such as Cirrhinus cirrhosus, Channa orientalis, Cyprinus carpio, and Wallago attu were available in the river. Among the identified native fish species, 52.73% of species showed decreasing population trends at the national level. Considering availability, the maximum fish species was rarely available (29.82%), followed by commonly available (28.07%), moderately available (22.81%), and abundantly available (19.30%). The main drivers affecting the river’s fish diversity were identified as indiscriminately overfishing, followed by dewatering fishing, dewatering for irrigation purposes, natural drought in the winter season, use of banned fishing gears, and katha fishing method. However, measures like the establishment and management of fish sanctuaries, creating alternate livelihood options for fishers, and building public awareness about fishing laws and regulations could be helpful in conserving the fish biodiversity of the Gurukchi River.