The modification of river flow regimes poses a significant threat to the world’s freshwater ecosystems. Northern Australia’s freshwater resources, particularly dry season river flows, are being increasingly modified to support human development, potentially threatening aquatic ecosystems and biodiversity, including fish. More information is urgently needed on the ecology of fishes in this region, including their habitat requirements, to support water policy and management to ensure future sustainable development. This study used electrofishing and habitat survey methods to quantify the dry season habitat use of 20 common freshwater fish taxa in the Daly River in Australia’s wet-dry tropics. Of twenty measured habitat variables, water depth and velocity were the two most important factors discriminating fish habitat use for the majority of taxa. Four distinct fish habitat guilds were identified, largely classified according to depth, velocity and structural complexity. Ontogenetic shifts in habitat use were also observed in three species. This study highlights the need to maintain dry season river flows that support a diversity of riverine mesohabitats for freshwater fishes. In particular, shallow fast-flowing areas provided critical nursery and refuge habitats for some species, but are vulnerable to water level reductions due to water extraction. By highlighting the importance of a diversity of habitats for fishes, this study assists water managers in future decision making on the ecological risks of water extractions from tropical rivers, and especially the need to maintain dry season low flows to protect the habitats of native fish.