The utility of artificial and natural substrate samplers affects the distribution of macro invertebrates in freshwater ecosystems. This study evaluated the efficiency of litter substrates of Croton macrostachyus, Eucalyptus grandis, Ficus sure, and Salix subserrata and artificial substrates/ bricks in terms of macro invertebrates colonization, abundance, family richness, uniformity, and composition of the sampled macro invertebrates. Few researchers have evaluated the efficiency of artificial and natural substrates in the colonization and dispersal of macro invertebrates in freshwater ecosystems. The objectives of this assessment were to evaluate the efficiency of artificial and natural substrate samplers in collecting macro invertebrates for water quality assessments of the Gilgel Gibe Watershade and its tributaries. Five sampling areas were chosen to compare macro invertebrates colonization in relation to the effect of inorganic (bricks) and leaf litter as substrates. Four organic or bedding substrates were selected and collected from the upper and lower study sites based on native and exotic plant species and their diversity along the study sites. Therefore, the following litter species were collected: Croton macrostachyus, Eucalyptus grandis, Ficu ssure, and Salix subserrata. After 30 days, all samples from each site were collected. Past software was used for descriptive and diversity indices analysis. A total of 5491 macro invertebrates individuals were counted on both substrates (artificial/bricks and natural/litter species). The compositions of Ephemeroptera and Trichoptera differed by litter substrates and were consistently higher in litter bags containing artificial substrates. In general, the family richness, uniformity, and composition of the artificial substrate samplers for macro invertebrates were higher than those of the litter substrates, which may indicate that the artificial substrates was more efficient than the litter substrates in dispersing macro invertebrates due to its ability to allocate habitat support to macro invertebrates.