Fish biodiversity assessments are vital for understanding threats and aquatic ecosystem health. In the Poso River, Central Sulawesi, changes in biodiversity are influenced by habitat alteration, non-native species, and overfishing. This study evaluates fish and crustacean biodiversity in the Poso River system to address these challenges for improving fisheries management, conservation, and fish passage integration into hydropower development. The present biodiversity study utilized a comprehensive methodology that encompassed both traditional taxonomic approaches and DNA barcoding, specifically targeting the mitochondrial cytochrome c oxidase subunit-1 (COI) gene for accurate species identification and spatial boundary validation. The survey was conducted in the upstream, hydropower area and downstream areas of the river. We found 27 species of fish in the Poso River, including both native (e.g., Anguilla celebesensis, Oryzias nebulosus, Rhyacichthys aspro) and non-native species (e.g., Melanochromis auratus, Amphilophus trimaculatus, Oreochromis niloticus). Two International Union for Conservation of Nature (IUCN) red listed endangered species were also observed (e.g., Adrianichthys poptae and Mugilogobius sarasinorum). DNA barcoding was performed to examine species spatial boundaries. The fish population in the Poso River was dominated by non-native species, accounting for 85.70% of the total population. The upstream reaches had the highest fish abundance and diversity, while the downstream reaches had the lowest. There was, however, no significant difference in species richness and diversity across different locations and seasons. The dominance of non-native species in the Poso River necessitates the improvement of existing fishways within the hydropower dams potentially through the development of selective technology that can block the distribution of these non-native species. This research highlights the critical issue of non-native species proliferation and the potential threat they pose to native fish populations, providing valuable insights for conservation and management efforts in Indonesia and similar ecosystems worldwide.