The rivers of Southern Appalachia are among the most biologically diverse freshwater systems in the temperate zone and are home to numerous endemic aquatic organisms. Throughout the Central Appalachian ecoregion extensive coal mining and the associated pollution associated with these mines significantly degrades the water quality of river networks. The extensive surface coal mines in the region generate alkaline mine drainage that raises the pH, the salinity and the trace element concentrations in streamwaters. While previous regional assessments have focused primarily on quantifying the impact of mining on macroinvertebrate and fish communities, here we expand these assessments with a more comprehensive evaluation across multiple kingdoms of life (bacteria, algae, macro-invertebrates, all eukaryotes and fish) using high-throughput amplicon sequencing of environmental DNA. We collected water samples from 93 streams in Central Appalachia (West Virginia) along a gradient of mountaintop coal mining activities to assess how this intense land use change alters downstream water chemistry and consequential impacts on aquatic biodiversity. For each group of organisms, we identified the sensitive and tolerant taxa along this gradient and we calculated stream conductivity thresholds at which large synchronous declines in diversity were observed using the Threshold Indicator Taxa Analysis. We found that streams below mining operations presented large declines in diversity (- 18 to - 41%) and large shifts in community composition that were consistent across multiple taxonomic groups. Overall, large synchronous declines in bacterial, photoautotrophic and macroinvertebrate communities occurred at low levels of mining impacts (stream conductivity thresholds of 150 to 200 µS/cm), which are nearly half of the current EPA aquatic life benchmark of 300 µS/cm for Central Appalachia streams. This study shows that extensive coal surface mining activities lead to the extirpation of 40% of biodiversity from this wildlife refuge and corridor and that the current water quality criteria are likely not protective of many groups of organisms across the tree of life.