The rock-hosted subseafloor biosphere provides key insights into the limits and origins of life, yet it remains largely unknown due to limited access. Recently, IODP Expedition 399 provided unprecedented access to a 1,268-meter core from the upper mantle of the Atlantis Massif, primarily composed of serpentinized harzburgite. The abundance and composition of indigenous organisms, their metabolic capabilities, physiological activity, and the role of serpentinization in sustaining life are critical, yet unanswered questions. However, the extremely low biomass and high DNA adsorption capacity of these mantle rocks present significant challenges for DNA extraction and contamination control, limiting our exploration of the rock-hosted biosphere. In this study, we made notable progress by distilling and refining DNA extraction protocols. Using 16S rRNA gene amplicon and metagenomic sequencing, we specifically developed the quality control and decontamination workflow tailored to the unique complexities of low-biomass samples. In this context, we characterized candidate microbial residents within the rocks and fluids, including Campylobacteria, Aquificae, Dehalococcoidia, Bathyarchaeia, Hadarchaeia, Methanosarcinia, and Nitrososphaeria, with distinct phylogenies from those typically found in seawater and sediments. These putative microbial residents likely play key roles in mediating the carbon, nitrogen, and sulfur cycles between the mantle rocks and formation fluids. Our findings suggest the presence of a complex metabolic network capable of thriving in the mantle rocks under high-temperature, hydrogen-rich, and alkaline conditions, underscoring the adaptability of microbial life in extreme subsurface environments. These results contribute to a broader understanding of life’s resilience in the deep biosphere and offer new insights into the origins of life and the potential for extraterrestrial life.

Serpentinization,Mantle peridotites,Origin and evolution of life,Microbe-mineral Interactions