Dams constitute a major threat to aquatic ecosystems world-wide by modifying habitat and impairing opportunities for many freshwater fish. Subsequently, there can be a loss in fish diversity and change in the species distributions and population structure that ultimately contribute to changes in trophic structure and ecosystem function. In this study, stable isotope analysis (SIA) was used to compare trophic interaction and niche space of five fish species residing upstream and downstream of a significant barrier (Tallowa Dam) on the Shoalhaven River in south-eastern Australia. Significant reduction in niche space was found among predator/prey species residing in upstream habitat, implying limited dietary opportunity. Mixing calculations for Macquaria novemaculeata confirmed ontogenetic differences upstream and downstream of the dam. Causal mechanisms for this variability in trophic structure include habitat modification and/or discontinuity in river connectivity. SIA provides a useful tool for demonstrating the effect of barriers and/or impoundments on aquatic ecosystems, and for developing future monitoring programmes to evaluate restoration strategies.