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The McMurdo Dry Valleys of Antarctica present extreme environmental challenges. Life is restricted to patchy occurrence of lichens, mosses and invertebrates, plus soil microbial communities. Fungi have been described in lichen symbioses but relatively little is known about the occurrence of free-living soil fungi in the Dry Valleys. A challenge in estimating fungal species richness has been the extent to which estimates based on either cultivation or environmental DNA reflect the active assemblage in cold-arid soils. Here, we describe analysis for inland Dry Valleys soil of environmental DNA and RNA (cDNA) to infer total and putative metabolically active assemblages, respectively, plus cultivation approaches using a variety of laboratory growth conditions. Environmental sequences indicated a highly restricted assemblage of just seven phylotypes that affiliated phylogenetically within two known genera, Helicodendron and Zalerion, plus previously unidentified fungal phylotypes. None of the commonly encountered molds and mitosporic genera recorded from maritime Antarctic locations were encountered. A striking difference was observed in the frequency of recovery for phylotypes between libraries. This suggests that both species richness and beta diversity estimates based on DNA libraries have the potential to misinform putatively active assemblages. Cultivation yielded a cold-tolerant Zalerion strain that affiliated with DNA and RNA library clones, and a psychrotrophic yeast (Debaryomyces hansenii), which was not detected using either culture-independent approach.