Determining the source and flow of carbon, energy and nutrients through food webs is essential for understanding ecological connectivity and thus determining the impact of management practices on biodiversity. We combined DNA sequencing, microarrays and stable isotope analyses to test whether this approach would allow us to resolve the carbon flows through food webs in a weir pool on the lower Murray River, a highly impacted, complex and regulated ecosystem in southern Australia. We demonstrate that small fish in the Murray River consume a wide range of food items, but that a significant component of carbon and nitrogen entering the food web during dry periods in summer, but not spring, is derived from nonconventional sources other than in-channel primary producers. This study also showed that isotopic analyses alone cannot distinguish food sources and that a combined approach is better able to elucidate food-consumer dynamics. Our results highlight that a major river ecosystem, stressed by reduced environmental flows, can rapidly undergo significant and previously undetected changes that impact on the ecology of the system as a whole.