Culture-independent molecular techniques enable us to analyze microbial communities in various environments. Molecular methods can detect numerous uncultivable prokaryotes in extreme environments including anaerobic, no-light, high-pressure, and high-temperature conditions. Recently, microbial eukaryotes were detected in the deep-sea environments, suggesting that microbial eukaryotes can adapt to a wider range of conditions than previously thought. At the Horonobe Underground Research Laboratory of Horonobe, Japan, we performed a culture-independent analysis of eukaryotes acquired at depths of −250 m and −270 m. Our results indicated that fungi are the dominant eukaryotic component in the deep sedimentary rocks of Horonobe. In the rock matrices, we detected a wide taxonomic range of fungi including Cryptomycota, Basidiomycota, and Ascomycota. This study is the first report on the diversity of eukaryotic species in deep terrestrial subsurface sedimentary rock matrices.