Biodiversity conservation has focused on large vertebrates and plants with pronounced economic value, compared with smaller organisms such as benthic macroinvertebrates that play a particular role in freshwater ecosystems. The increasing loss and degradation of freshwater habitats, coupled with low taxonomic and ecological knowledge and limited conservation efforts in the tropical regions, threaten macroinvertebrate biodiversity. This study investigated spatial and seasonal variability in benthic macroinvertebrate diversity and the underlying environmental variables within the Aturukuku River in Tororo, Uganda. Benthic macroinvertebrates were evaluated by comparing their composition at a reference site in the upstream rural area with two urban midstream sites and one rural downstream site during dry and wet seasons in 2018. Benthic macroinvertebrate diversity in the Aturukuku River was lower than that reported from least disturbed small rivers within the same region; with moderate to high pollution tolerant taxa, dominating. Chironomidae, Oligochaeta and Simulium sp. predominated at the urban sites receiving sewage and abattoir effluent. The Shannon–Wiener diversity index, species richness and species evenness in the river were generally low. Prosobranch (Thiaridae) snails and bivalves (Sphaeridae) were among the taxa with low tolerance to changes in the river. Canonical Correspondence Analysis revealed that total nitrogen, dissolved oxygen, temperature and streambed substrate were predominant in structuring benthic macroinvertebrate assemblages. There was no potential species-rich area for macroinvertebrate conservation, given the poor environmental conditions and diversity in the entire river. However, specific mitigation measures against the declining quality of the river are recommended, to boost conservation of macroinvertebrates and the entire riverine ecosystem.