The deep sea is the largest biosphere on Earth and a key frontier in ecological research. The subseafloor, composed of diverse and largely unexplored environments, harbors abundant microbial life. Despite limited access, it is estimated that around 80% of Earth’s microbial biomass resides in the deep subsurface, making it one of the planet’s largest microbial reservoirs. These environments are particularly important such as marine sediments acting as major carbon sinks and associated microbial activity playing a crucial role in global biogeochemical cycles. However, the diversity and ecology of microorganisms, particularly microbial eukaryotes, remain critically understudied due to the scarcity of cultured representatives and genomic data. Current knowledge relies heavily on metabarcoding, which lacks resolution due to limited reference databases and the high proportion of unknown and undescribed taxa. This project addresses these critical knowledge gaps through an integrative approach combining microbial cultivation and whole-genome sequencing to investigate subseafloor microbial eukaryotes. Our research addresses three principal scientific objectives: (1) characterizing the taxonomic diversity and phylogenetic relationships of subseafloor eukaryotes to reveal hidden biodiversity and evolutionary patterns; (2) elucidating their functional capabilities and metabolic strategies through genomic analyses to understand their roles in biogeochemical processes; and (3) investigating their potential ecology within these extreme environments. This research aims to advance our current understanding of subseafloor ecosystems and their roles in global biogeochemical cycles, with implications for biology, ecology, evolution, and biotechnology, ultimately illuminating one of Earth's most enigmatic and significant microbial frontiers.