The freshwaters of the southern Gulf of Carpentaria region is a poorly researched area of Australia, based on western science approaches, but rich in aquatic life. First Nations people have been an integral part of this region for tens of thousands of years, and have a deep understanding of the species and habitats. This study surveyed freshwater fish and other aquatic animals across more than 30 aquatic systems, to assess the species diversity, abundance and life history information. This information was combined with water quality and habitat condition information to determine potential threats to these species, including from water development. More than 34 species of fish, as well as turtles and crustaceans were identified across 30 river, creek, waterhole and wetland sites. There were distinct differences in fish dominance based on water quality and site characteristics, and proximity to the coast. Traditional knowledge of fish was combined with western science information to gain a more integrated picture of the cultural and environmental values. Fish species of particular significance as a food source were identified, as well as traditional practices of hunting and eating.