Deep subsurface microbiology is a rising field in geomicrobiology, environmental microbiology and microbial ecology that focuses on the molecular detection and quantification, cultivation, biogeographic examination, and distribution of bacteria, archaea, and eukarya that permeate the subsurface biosphere. The deep biosphere includes a variety of subsurface habitats, such as terrestrial deep aquifer systems or mines, deeply buried hydrocarbon reservoirs, marine sediments and the basaltic ocean crust. The deep subsurface biosphere abounds with uncultured, only recently discovered and—at best—incompletely understood microbial populations. So far, microbial cells and DNA remain detectable at sediment depths of more than 1 km and life appears limited mostly by heat in the deep subsurface. Severe energy limitation, either as electron acceptor or donor shortage, and scarcity of microbially degradable organic carbon sources are among the evolutionary pressures that may shape the genomic and physiological repertoire of the deep subsurface biosphere. Its biogeochemical importance in long-term carbon sequestration, subsurface elemental cycling and crustal aging, is a major focus of current research at the interface of microbiology, geochemistry, and biosphere/geosphere evolution. The papers of this Frontiers e-volume bear evidence of the rapid advances in deep subsurface microbiology.