Fish biodiversity assessments play a crucial role in identifying potential threats, and the overall health of aquatic ecosystems. Poso River in Sulawesi, Indonesia presents a complex scenario where changes in fish biodiversity can be influenced by habitat alteration, the introduction of non-native fish species and overfishing. In this study, we assessed fish biodiversity in Poso River to gain a better understanding of the challenges to its aquatic biodiversity. This knowledge is critical for enhancing fisheries management and conservation programs, and is essential for improving the fishway system integrated into hydropower dams. The 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 accurately identify species and validate their boundaries. It was conducted in upstream, environmental flows of hydropower dams, and downstream areas of the river. We found 27 species of fish in the Poso River, including both native and non-native species. Two endangered species were also observed. DNA barcoding was performed to examine species boundaries and identity. The fish population in the Poso River is dominated by non-native species, accounting for 85.70% of the total population. The upstream area had the highest fish abundance and diversity, while the downstream area had the lowest. There was 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 fish passages equipped in hydropower dams through the development of selective fish passages that can block the distribution of these invasive species. This research highlights the critical issue of non-native species proliferation and its potential threat they pose to native fish populations, providing valuable insights for conservation and management efforts in Indonesia and similar ecosystems worldwide.