The study of eukaryotic extremophiles is relatively novel, and, therefore, documentation of the structure and function of micro-organisms in continental hydrothermal systems globally is limited. In this study, we investigate fossil diatoms in siliceous hydrothermal deposits of the Upper Geyser and Yellowstone Lake hydrothermal basins in Yellowstone National Park, and utilize preserved diatom assemblages to infer local environmental conditions. Siliceous sinter from both the Upper Geyser Basin and Yellowstone Lake contains evidence of in-situ diatom growth within these environments. At Upper Geyser Basin, the assemblage consisted of species that could grow on moist siliceous sinter and was dominated by Rhopalodia gibberula. Diatom valves were found in various preservation states, ranging from nearly pristine to highly diagenetically altered. Diatoms collected from siliceous spires in Yellowstone Lake consisted largely of tychoplanktonic and benthic species that were almost certainly growing on the outside of the structure, with an assemblage indicative of relatively shallow, alkaline waters. What remains unclear without access to material for high-resolution dating is whether diatoms colonized the spires during hydrothermal activity or after activity ceased. Our results indicate that diatom frustules can, to some extent, survive alteration in low-temperature (<76°C) hydrothermal environments.