The Martuwarra–Fitzroy River is a 733-km-long, heritage-listed river in the Kimberley region of Western Australia. It supports exceptional ecological and cultural values but has faced proposals for large-scale water-resource development. To inform water planning and diverse stakeholder groups, we undertook a four-year research program examining how different components of the flow regime support ecological processes across the river and its floodplain. Our studies combined field surveys, remote sensing and hierarchical modelling to investigate main-channel and floodplain habitats, with responses ranging from the condition and growth of individual fish to species distributions and assemblage diversity. The results demonstrate that flow performs multiple, interconnected ecological functions. Wet-season flows replenish the energy reserves of culturally important fork-tailed catfish and promote the longitudinal and lateral connectivity needed to support the distribution and life-history requirements of migratory prawns. Although the floodplain is often inundated only briefly, it provides highly productive habitat where elevated zooplankton biomass enhances the growth and potential survival of young-of-year fish. When lateral connectivity is extensive and sustained, more fish access the floodplain, allowing this otherwise potentially risky habitat to function as an important nursery. Groundwater-fed pools in the main channel, particularly those that retain considerable depth throughout the dry season, provide critical refuges for the fish metacommunity, especially large-bodied species. Protecting the river’s biodiversity therefore relies on maintaining the seasonal sequence of hydrological processes that connect river habitats, inundate productive floodplains and sustain dry-season refuges. By linking ecological responses to measurable hydrological and hydraulic metrics, we produced empirical relationships that could be directly applied in water-resource planning. Our program provides an example of transparent knowledge generation and equitable knowledge sharing. Ultimately, the decision was made not to allocate surface water from the Martuwarra–Fitzroy River. This research was funded by the Australian Government’s National Environmental Science Program.