Despite significantly influencing fisheries characteristics of major global basins, medium-sized rivers such as Awach Kibuon remain the least studied. This study evaluated longitudinal fish species diversity, distribution, and environmental influence in Awach Kibuon River, Lake Victoria Basin, Kenya, to inform sustainable management of fisheries resources within the region. Nine sites located in different zones of the river were sampled monthly for eight months in 2019 and 2020. Fish samples were collected for 45 minutes using a backpack electrofisher on a 50-m river reach in every sampling site. Selected physico-chemical parameters (pH, total dissolved solids (TDS), dissolved oxygen (DO), temperature, conductivity, and turbidity) were measured in situ using standard methods. Longitudinal river fish diversity was assessed using species richness, Simpson's Index (D), Shannon-Weiner Index (H′), and Pielou's Evenness Index (J). A total of 983 fish specimens belonging to 21 species, 12 genera, seven families, and six orders were caught during sampling. Cyprinidae was the most dominant family, represented by nine species, followed by Claridae, Mochokidae, and Cichlidae, represented by three species each. Longitudinal species richness, abundance, and diversity indices deviated from the expected downstream increase. Upstream stations had no fish, whereas midstream and river mouth stations had moderate and high species diversity, respectively. Pearson's correlation analyses showed that temperature, conductivity, TDS, depth, and velocity influenced fish species richness in the river. The above findings call for the inclusion of medium-sized rivers in fisheries management plans of Great Lakes Basins for sustainability.