The oceanic and continental lithosphere constitutes Earth’s largest microbial habitat, yet it is scarcely investigated and not well understood. The physical and chemical properties here are distinctly different from the overlaying soils and the hydrosphere, which greatly impact the microbial communities and associated geobiological and geochemical processes. Fluid–rock interactions are key processes for microbial colonization and persistence in a nutrient-poor and extreme environment. Investigations during recent years have spotted microbial processes, stable isotope variations, and species that are unique to the subsurface crust. Recent advances in geochronology have enabled the direct dating of minerals formed in response to microbial activity, which in turn have led to an increased understanding of the evolution of the deep biosphere in (deep) time. Similarly, the preservation of isotopic signatures, as well as organic compounds within fossilized micro-colonies or related mineral assemblages in voids, cements, and fractures/veins in the upper crust, provides an archive that can be tapped for knowledge about ancient microbial activity, including both prokaryotic and eukaryotic life. This knowledge sheds light on how lifeforms have evolved in the energy-poor subsurface, but also contributes to the understanding of the boundaries of life on Earth, of early life when the surface was not habitable, and of the preservation of signatures of ancient life, which may have astrobiological implications. The Special Issue “Tracking the Deep Biosphere through Time” presents a collection of scientific contributions that provide a sample of forefront research in this field. The contributions involve a range of case studies of deep ancient life in continental and oceanic settings, of microbial diversity in sub-seafloor environments, of isolation of calcifying bacteria as well as reviews of clay mineralization of fungal biofilms and of the carbon isotope records of the deep biosphere.