In this study, the composition and structure of the macrobenthic community in the Ulungu River Basin was investigated and the water quality status of the basin was evaluated. In May and August 2022, and October 2023, the macrobenthic and water environmental factors at 11 sample sites in the basin were investigated, and the water quality of the basin was evaluated by the Shannon–Wiener index, FBI, and BMWP index. A total of 6101 macrobenthic organisms were collected; these organisms belonged to 3 phyla, 7 classes, 14 orders, 57 families, and 117 genera. Arthropod species accounted for the largest number of species (87.9%). A total of nine dominant species were found: Micronecta sp., Eukiefferiella sp., Baetis sp., Polypedilum sp., Saetheria sp., Ephemerella sp., Limnodrilus sp., Ephemera sp., and Hydropsyche sp. At the temporal level, the average density and biomass of macrobenthos were in the order of August > October > May; at the spatial level, the average density was greater in the tributaries than in the main stream, and the average biomass was greater in the main stream than in the tributaries. The mean values of the Shannon—Wiener index and Margalef richness index were as follows: August > October > May at the temporal level; the mean values of the Pielou evenness index were as follows: May > October > August at the temporal level. At the spatial level, the overall mean value of each diversity index indicated that the tributaries had larger diversity indices than the main stream. The water quality evaluation results revealed that the overall water quality level of the Ulungu River Basin ranges from light pollution to poor quality. Human interference activities greatly impact the water quality of the basin. To restore the ecology of the basin, it is necessary to strengthen the management and control of pollution sources.