Worldwide, freshwater systems contain more than 18,000 fish species^1,2,3, which are critical to the functioning of these ecosystems⁴ and are vital cultural and economic resources to humans^5,6,7; despite this value, fish biodiversity is at risk globally^8,9. In the USA, leading threats to fish communities in rivers and streams include climate change and invasive fish introductions and game fish stocking by humans^{10,11,12,13,14}. Here we harmonized US federal biomonitoring datasets with 389 species spanning 27 years (1993–2019) and 2,992 sites to analyse trends in fish biodiversity. In cold streams (past summer stream temperatures below 15.4 °C), fish abundance and richness declined by 53.4% and 32% over 27 years, respectively, and uniqueness increased. Periodic (large-bodied, late-maturing) fishes increased, and opportunists (small-bodied, short generation time, ‘r-selected’) decreased, possibly due to proliferation of native or introduced game fishes. In warm streams (stream temperatures greater than 23.8 °C), fish abundance and richness increased by 70.5% and 15.6% over 27 years, respectively, and communities homogenized. Small opportunistic fishes replaced large periodic fishes. Intermediate streams (stream temperatures 15.4–23.8 °C), representing the average stream, had minimal changes in fish biodiversity through time. Interactions between warming and introduced fish were associated with increased rates of degradation to local fish biodiversity. Given the magnitude of these changes in a relatively short time span, there is an urgent need to curb degradation of fish biodiversity caused by fish introductions and warming water temperatures.