Food web studies integrate ecological information and provide understanding of ecosystem function. Aquatic ecosystems of the Kimberley region (north-Western Australia) have high conservation significance as hotspots for maintaining local and regional biodiversity. This study investigated the influence of waterhole type and persistence on the strength of consumer reliance on local energy resources for aquatic food webs. Changes in water isotopic composition indicated groundwater inputs were enough to overcome evaporative losses in some waterholes. Other waterholes had varying levels of isotope enrichment suggesting insufficient groundwater input to 'compensate' for evaporative loss. C and N isotope analysis indicated considerable overlap among energy sources in waterholes between macrophytes and periphyton but gradient analysis indicated that periphyton is a major carbon source for aquatic consumers. Groundwater-fed waterholes appeared to have higher quality food sources (indicated by lower C:N ratios), but there was minimal evidence that direct groundwater contributions were related to food web processes. Nonetheless, in a region where groundwater is influential in maintaining aquatic habitats, future development of groundwater reserves will likely affect the ecological and cultural value of freshwater wetlands by either reducing their permanence or size or indirectly through possible alteration to the role of periphyton in supporting the food web.