Ecological control of blackflies (Simulium damnosum) can be an alternative or additional tool to enhance onchocerciasis elimination efforts. However, limited research is conducted on the ecology of blackflies in Ethiopia. In this study, we determined the habitat preference of blackfly larvae and their relationship with aquatic macroinvertebrate predators in the Omo Gibe river basin of southwest Ethiopia. Environmental and biological data were collected from 150 sampling sites during both dry and wet seasons in 2019. Generalized Linear Models (GLMs) were used to identify factors affecting the occurrence and abundance of S. damnosum larvae. Canonical Correspondence Analysis (CCA) was used to investigate the relationship between environmental and biological variables and the abundance of S. damnosum larvae. The findings of this study indicated the abundance of S. damnosum larvae increased with increasing turbidity, alkalinity and altitude, but decreased with increasing concentrations of five-day Biological Oxygen Demand (BOD5), orthophosphate and magnesium ion. Both the presence and abundance of S. damnosum larvae decreased with the increasing abundance of stonefly larvae (Perlidae). Simulium damnosum larvae were found less likely in the presence of mayfly larvae (Baetidae) and were less abundant where Chironomidae are abundant. In conclusion, the findings of this study showed that the habitat preference of S. damnosum larvae is determined by environmental factors and that the presence and abundance of the larvae are affected by macroinvertebrate predators. It is essential to establish buffer zones as a part of watershed management to retain pollutants and prevent them from entering directly into water courses to improve water quality and the assemblages of macroinvertebrate predators and enhance biocontrol of blackflies.