The subsurface environment of Earth contains a diverse and complex community comprised of >1028 microbial cells globally, with a significant portion of this biomass being present in continental margin sediment. The cellular functions, and the genes that encode them, enabling subsurface microbial survival over geological timescales are unknown. To better understand the cellular functions that enable microbial survival over geological timescales in subseafloor sediment, metatranscriptomes from subseafloor continental margin sediment (5–159 m below the seafloor) were compared to soil metatranscriptomes using fungal genomes from the genera Cryptococcus and Aspergillus, taxa known to occupy representative samples of each environment, as reference. Soil metatranscriptomes contain a relatively higher number of overexpressed representative fungal homologous genes involved in catabolism of labile substrates, reflecting the increased bioavailability of the soil substrate pool relative to subseafloor sediment. In contrast, many fungal homologs with overexpression in subseafloor samples encode proteasomes and autophagosomes that likely help conserve and recycle amino acids under reduced availability of labile organic matter. Genes associated with stationary phase were significantly overexpressed in the subseafloor suggesting a relatively higher investment into cellular maintenance energy. Such differences indicate that 1.) subseafloor fungal transcripts are not contaminants and 2.) subseafloor fungi are relatively dormant compared to soil fungi, and likely persist for long periods in stationary phase. Our findings provide insights into biochemical mechanisms enabling subseafloor survival for long periods, under extreme pressure, with relatively recalcitrant carbon sources.