The Vestfold Hills are a 400 km2, isolated ice-free oasis in eastern Antarctica featuring large areas with translucent quartz rocks that provide habitat for hypolithic microbial communities underneath. We used high-throughput DNA sequencing of 16S and 18S ribosomal RNA amplicons to characterize bacterial and eukaryotic hypolithic communities across the Vestfold Hills. We found high-level, local heterogeneity in community structure consistent with limited dispersal between hypoliths. Hypolithic communities were dominated by heterotrophic Bacteroidetes (mean bacterial relative read abundance: 56%) as well as Cyanobacteria (35%), with the eukaryote component often dominated by Chlorophyta (43%). Small but significant proportions of the variation in microbial community composition and function were explained by soil salinity (5–7%) and water availability (8–11%), with distinct taxa associated with different salinities and water availabilities. Furthermore, many inferred bacterial metabolic pathways were enriched in hypolithic communities from either dry or high-salinity sites. Vestfold Hills hypolithic habitats are likely to be local refuges for bacterial and eukaryotic diversity. Gradients in soil salinity and water availability across the Vestfold Hills, in addition to the number and diversity of lake types and fjords as potential source populations, may contribute to the observed variation in the extremophile, hypolithic microbial community composition.