Surveys of fish in the mid-Goulburn River, between Lake Eildon to Lake Nagambie, have shown the native and freshwater fisheries in this reach are sub-optimal despite there being good habitat. Macroinvertebrates have also been shown to be in poor condition. A major contributing factor to poor fish and macroinvertebrate community health in the region may be from the water releases from Lake Eildon. The large volumes of water released from Lake Eildon have significantly altered the flow regime of the river, and provide unseasonably cold waters during spring and summer that impact the river as far downstream as Seymour. The study aimed to determine whether the abundance of macroinvertebrates was low where there were low numbers of fish and to determine whether they were stressed from the modified flow regimes and associated water quality caused by Lake Eildon. Macroinvertebrate community was assessed at four sites within the mid-Goulburn and King Parrot Creek over multiple levels of organisation (community, population, individual and sub-lethal) in a multiple lines of evidence approach. Several methods were employed across three seasons (spring, summer and autumn) including replicated edge sweep samples (community structure, relative abundance of key taxa, biomass), artificial substrate samples (community structure, relative abundance of key taxa, biomass) and shrimp (Paratya australiensis) cage studies (survival, growth, biomarkers). Our results show that macroinvertebrate fauna in the Goulburn River are impaired and very different to those in King Parrot Creek. A lack of large bodied crustaceans (shrimp) is evident in the mid-Goulburn despite surrounding tributaries containing high abundances of them. It is likely that large water releases from Lake Eildon are impacting macroinvertebrates in the Goulburn River, with the greatest impacts occurring in spring and summer when water temperatures associated with the water releases were colder. The management implications of these findings will be discussed.