Deposits of fly ash and other coal combustion wastes are common remnants of the energy industry. Despite their environmental risks from heavy metals and trace elements, they have been revealed as refuges for threatened terrestrial biodiversity. Surprisingly, freshwater biodiversity of fly ash sedimentation lagoons remains unknown despite such lack of knowledge strongly limits the efficient restoration of fly ash deposits. We bring the first comprehensive survey of freshwater biodiversity, including nekton, benthos, zooplankton, phytoplankton, and macrophytes, in fly ash lagoons across industrial regions of the Czech Republic. To assess their conservation potential, we compared their biodiversity with abandoned post-mining ponds, the known strongholds of endangered aquatic species in the region with a shortage of natural ponds. Of 28 recorded threatened species, 15 occurred in the studied fly ash lagoons, some of which were less abundant or even absent in the post-mining ponds. These are often species of nutrient-poor, fishless waters with rich vegetation, although some are specialised extremophiles. Species richness and conservation value of most groups in the fly ash lagoons did not significantly differ from the post-mining ponds, except for species richness of benthos, zooplankton, and macrophytes, which were slightly lower in the fly ash lagoons. Although the concentrations of some heavy metals (mainly Se, V, and As) were significantly higher in the fly ash lagoons, they did not significantly affect species richness or conservation value of the local communities. The differences in species composition therefore does not seem to be caused by water chemistry. Altogether, we have shown that fly ash lagoons are refuges for threatened aquatic species, and we thus suggest maintaining water bodies during site restoration after the cessation of fly ash deposition. Based on our analyses of environmental variables, we discuss suitable restoration practices that efficiently combine biodiversity protection and environmental risk reduction.