The rivers of northern Australia are some of the most un-impacted river systems in the world. These rivers also support Australia’s most species rich freshwater fish assemblages, however basic biological knowledge of many species is limited. The Daly River is a perennial river system in the Northern Territory that supports a highly diverse native freshwater fish fauna, important recreational and commercial fisheries, and provides significant cultural value to its Indigenous people. This study examines the patterns of habitat use of several native fish species in the Daly River, by modelling their habitat use in the early and late dry season using a comprehensive 10-year dataset. Data were analysed using Boosted Regression Trees modelling and multivariate analysis approaches. Results highlight the importance of key habitat types in the river for various species, and include parameters such as water velocity, depth and substrate type. These findings will result in a better understanding of fish habitat use for a large number of species, and will contribute to better informed management activities such as environmental water protection to protect important fish habitats.