Rivers around the world are threatened by altered flow due to water resource development. Altered flow can change food webs and impact riverine energetics. The Fitzroy River, in northern Australia, is targeted for development but uncertainty remains about the sources of carbon supporting the food web, particularly in the lowlands-the region most likely to be impacted by water extraction. This study used stable isotopes to investigate if algal biofilm is the main carbon source sustaining fish in lowland habitats. We also sought evidence that large-bodied migratory fish were transporting remote carbon around the system. Our results revealed that local algal biofilm carbon was the dominant source of energy sustaining fish in wet season floodplain habitats, but that fish in main-channel pools during the dry season were increasingly dependent on other carbon sources, such as leaf litter or phytoplankton. We found no evidence that large-bodied fish were transporting remote carbon from the floodplain or estuary into the lower main-channel of the river. We recommend that water planners take a precautionary approach to policy until sufficient food web evidence is amassed. Freshwater ecosystems sustain remarkable biodiversity and provide important services to nature and society but are degrading globally at an increasing rate 1. A common threat to freshwater systems is changes in flow-regime arising from water resource development to support agriculture 2,3. Changes in flow regime can alter hydrological connectivity between the river and its estuary, the river and its floodplain, and between surface and subsurface water 2,4,5. Altered connectivity may reduce the movement of nutrients and animals, and change the flow of energy through the food web, ultimately reducing the abundance of species valued by people (e.g. fish) 6,7. Understanding how riverine food webs function is essential if we are to estimate the likely impacts of flow alteration, make informed water management decisions and enact effective policy. The implementation of environmental flows policy is now considered critical to reverse the decline of freshwater biodiversity 8. There is an increasing focus on water management decisions and environmental flow policy in the wet-dry tropics of northern Australia, as pressure mounts to develop the region's large river systems 9,10. The region includes some of Australia's most intact freshwater ecosystems, which support high biodiversity, numerous endemic species and many fish which move between riverine, floodplain and estuarine environs 11. The Fitzroy River, in the Kimberley region of northwestern Australia, has been identified as having potential for development 10 , and at least one application to divert water from the river to grow fodder crops for cattle is being considered by government. This intermittent river, 700 km in length 10 , is highly valued by the Indigenous peoples who live along it, and for whom customary harvest of large-bodied fish and freshwater prawns is important 12. Water managers are tasked w...