We assessed whether tributaries in upland catchments (=watersheds) affected assemblages of benthic macroinvertebrates in mainstems, as has been reported in northern hemisphere systems. Eight confluences of small to medium streams (stream orders 1–4, 2.2–10.8 mwide) were studied in the Acheron River basin in Victoria, Australia. For each confluence, two transects were sampled at each of five zones relative to the confluence: two zones upstream in the mainstem, one zone upstream in the tributary, one zone at the confluence and one zone downstream in the mainstem. Surveys were conducted in both high-flow and low flow conditions. In mainstems, there was no change in macroinvertebrate density, taxonomic richness or functional feeding group composition downstream relative to upstream of the confluences. While tributaries statistically had distinctive benthic macroinvertebrate assemblages compared to mainstems, these distinctions were small. In low flows, densities in tributaries were substantially lower than in mainstems, but densities during high flows were more similar (albeit only about one-third as high as in low flow) in tributaries and mainstems. An inverse pattern was evident for taxonomic richness, where richness in tributaries and mainstems was similar in low flows but was greater in mainstems than in tributaries in high flows. We found little evidence of tributary effects in macroinvertebrate assemblages in this basin, which is at odds with some previous results from other continents. To explain this divergence, we suggest a conceptual model outlining factors that control variation in effects of tributaries on assemblages of benthic macroinvertebrates in mainstems.