Meeting the conservation challenges of long‐lived animal species necessitate long‐term assessments of trophic ecology. The use of dietary proxies, such as ratios of naturally occurring stable isotopes in animal tissues demonstrating progressive growth, has shown considerable promise to reconstruct trophic histories of long‐lived organisms experiencing environmental change. Here, we combine innovative radiocarbon scale‐ageing techniques with stable isotope analysis of carbon and nitrogen from cross sections of scale to reconstruct the trophic ecology of Australian lungfish (Neoceratodus forsteri) across its remaining global distribution. Over a 65‐year period, we found pronounced temporal shifts in the δ13C and δ15N isotopic ratios of lungfish that coincided with a period of hydrological modification by dams and land‐use intensification associated with agriculture and livestock grazing. In the Brisbane and Burnett Rivers, whose hydrology is substantially regulated by large dams, lungfish showed consistent trends of δ13C depletion and δ15N enrichment over time. This may indicate anthropogenic changes in background isotopic levels of basal energy sources and/or that additional seston exported downstream from impoundments represent a carbon source that was previously unavailable, thus shifting lungfish diet from benthic‐dominated primary production typical of unmodified river systems, to pelagic carbon sources. By contrast, δ13C ratios of lungfish in the unregulated Mary River were more stable through time, whereas δ15N ratios increased during a period of dairy industry expansion and increased application of nitrogen fertilization and then subsequently decreased at the same time that rates of pasture development declined and nutrient inputs presumably decreased. In conclusion, we provide evidence for human‐caused alterations in background isotopic levels and potential changes in availability of benthic versus pelagic energy resources supporting Australian lungfish and demonstrate how detectable trophic signals in long‐lived fish scales can reveal long‐term anthropogenic changes in riverine ecosystems.