Accurate data on the distribution patterns of fish species in aquatic ecosystems can be challenging to obtain using traditional methods. Environmental DNA (eDNA) analysis is a non-invasive technique that has demonstrated higher sensitivity and efficiency than conventional detection approaches in aquatic environments. In this study, we assessed the efficacy of eDNA compared to three standard survey methods—gill nets, electrofishing, and fyke nets—in detecting an endangered native species, Macquaria australasica (Macquarie perch), and an abundant alien species, Oncorhynchus mykiss (rainbow trout), within the Cotter River, a tributary of the Murrumbidgee River in the Murray–Darling Basin. The Cotter River supports one of the few remaining self-sustaining populations of M. australasica. Occupancy estimates showed that eDNA was a more sensitive and efficient detection method than each traditional method used individually and in combination. eDNA analysis revealed broader distribution of O. mykiss across all sampled sites, while M. australasica was detected at fewer locations and at lower concentrations. Notably, eDNA was able to detect M. australasica at low densities at a translocation site and an additional upstream location, indicating potential range expansion. These findings demonstrate that eDNA outperforms established survey techniques for both rare and common freshwater fish species and can provide valuable insights for conservation and management.