The deep biosphere of subseafloor basalts is thought to consist of mainly prokaryotes (bacteria and archaea). Here we report fossilized filamentous microorganisms from subseafloor basalts interpreted as fossilized fungal hyphae, probably Dikarya, rather than fossilized prokaryotes. The basalts were collected during the Ocean Drilling Program Leg 197 at the Emperor Seamounts, North Pacific Ocean, and the fossilized fungi are observed in carbonate-filled veins and vesicles in samples that represent a depth of ∼150 m below the seafloor. Three-dimensional visualizations using synchrotron-radiation X-ray tomographic microscopy show characteristic fungal morphology of the mycelium-like network, such as frequent branching, anastomosis, and septa. Possible presence of chitin in the hypha walls was detected by staining with Wheat Germ Agglutinin conjugated with Fluorescein Isothiocyanate and examination using fluorescence microscopy. The presence of fungi in subseafloor basalts challenges the present understanding of the deep subseafloor biosphere as being exclusively prokaryotic.