The majority of rivers in the Australian tropics possess near-natural flow regimes and are an ecological asset of global significance. We examined flow variability in large floodplain rivers in the Gulf of Carpentaria, northern Australia, and the potential ecological impacts of future water resource development (WRD). Flow metrics based on long-term records were used to classify flow regimes and predict hydrological drivers of ecological function. Flow regimes of selected rivers were then compared with those simulated for pre- and post-WRD flows in the Darling River, a highly modified river in Australia's south-east. Generally, rivers were classified as typically 'tropical' (more permanent, regular flows) or 'dryland' (more ephemeral, with greater flow variability). In addition, all rivers displayed wet-dry seasonality associated with changes in flow magnitude or number of zero-flow days. We propose that these features (flow permanence and regularity; flow variability and absence; wet-dry seasonality) are the key hydrological drivers of biodiversity and ecological function in the floodplain rivers of Australia's north. In terms of WRD, inter-annual flow variability was predicted to increase or decrease depending on rivers' natural flow regimes, specifically their tendency toward lower or higher flow magnitudes. Either outcome is expected to have adverse effects on the biodiversity and ecological function of these relatively pristine rivers and floodplain habitats. In particular, reduced and homogenised habitat, loss of life-history cues, inhibited dispersal and shifts in community composition, as a result of WRD, threaten the ecological integrity of rivers adapted to the three hydrological drivers above. These findings serve as a caution for careful consideration of water resource development options for rivers in the Australian tropics and for those with similar flow regimes the world over.