Human population growth in a changing climate and subsequent increase in water demands have resulted in proposals for many dams to be built, enlarged or refurbished. Recently, the value of reservoirs as refuges for threatened fish species has been highlighted, but the trophic and biotic interactions that occur in reservoirs need to be understood if they are going to be successfully managed as fish refuges. Recently Cotter Dam, in south-eastern Australia, was enlarged to twenty times its previous capacity. This offered a unique opportunity to investigate changes in water quality, invertebrates, fish diet and food web as the reservoir was in its initial filling stage. Sampling occurred in the early to mid-stage of the reservoir filling. Water quality data, in particular nitrogen and phosphorus, were collected in autumn and spring, with supplementary data supplied by the water owner. Micro- and macro- invertebrates were sampled in autumn and spring, along with Macquarie perch (Macquaria australasica) and rainbow trout diet (Oncorhynchus mykiss) and stable isotope sampling (δ13C and δ15N). We found that, during the early filling stage, nutrients in the reservoir increased, as did microinvertebrates. Macroinvertebrates remained stable in the initial filling stages. Initially, fish diet changed from aquatic invertebrates to terrestrial items, particularly terrestrial oligochaetes. By mid-filling there was a shift back to aquatic invertebrates and an increase in piscivory in trout. Stable isotope analysis showed how the importance of terrestrial inputs changed throughout the filling stage. Understanding the impact that enlargement of reservoirs has on fish diet and prey availability is important, as changes in food availability may affect fish condition, and potentially their reproductive output. Knowledge gained from this project may be used to enhance future management decisions surrounding reservoir filling and its implications for native freshwater fish species.