Dominant ecological paradigms provide theoretical frameworks for two basal sources of carbon to riverine food webs: carbon fixed by algae and submerged macrophytes (autochthonous) and carbon fixed by terrestrial vascular plants that enters aquatic ecosystems as litter (allochthonous). An additional, less studied, pathway for carbon to enter rivers is as methane that is produced in small quantities via methanogenesis by anaerobic methanogenic archaea. Because rivers are generally well oxygenated, methane is not often recognised as a substantial contributor to riverine carbon budgets. However, methane may also enter rivers via natural gas macro–seeps where underground deposits connect to the surface via fissures and fractures. We explored the contribution of methane to food webs by comparing δ13C values of animals and their foods from reaches rich in dissolved biogenic methane with reaches where methane concentrations were at background levels. Animals collected from natural gas macro–seeps zones had significantly depleted δ13C values compared to animals from reference zones. Within natural gas macro–seep zones, methane served as the principal basal carbon source for mayfly primary consumers (Tasmanocoenis spp.). We demonstrate the persistence of methane–derived carbon across multiple trophic levels of the natural gas macro–seep food web, subsidising >20% of the carbon in predatory fish. Carbon of terrestrial origin dominated biomass contributions to River prawns (Macrobrachium australiense) and Shrimp (Parataya australiensis), while autochthonous carbon contributions were generally lower (≤ 20%). Our findings establish the prevalence of a natural gas derived methane–methanotrophic bacteria–primary consumer pathway and underscore its significance as a potentially dominating energy conduit in rivers with high concentrations of dissolved methane. Additionally, we provide useful information for the incorporation of different sources of carbon in riverine food webs.