Evidence for the decline in native fish communities in the lowland Goulburn Catchment has been based primarily on studies investigating changes to environmental conditions as a result of river regulation. The present study aggregates historical fisheries data to spatially compare native fish communities in different parts of the catchment. Multivariate analyses demonstrate that distinctly different community compositions are found in different parts of the catchment. Eildon Dam, a deep release reservoir, has drastically altered the hydrology and water quality of the upper Goulburn River, and ordination analyses show that this has created both physical and artificial barriers to movement of native fish into the main channel. Concurrently, fisheries data from the upper main channel show the community is highly divergent compared to the remaining catchment. Further downstream, Goulburn Weir creates a barrier to fish movement between Lake Nagambie and the lower Goulburn Catchment, and alters the natural hydrological regime. Similarity Percentage analyses demonstrate that these stressors have facilitated the establishment of alien species, which dominate fish abundance in the upper and lower main channels. The current study emphasises the need to explore alternatives for improving management strategies to rehabilitate fish communities throughout the Goulburn Catchment.