The microbial biosphere in serpentinizing subseafloor rocks is globally significant. Tantalizing evidence from studies of the Lost City Hydrothermal Field and continental ophiolites indicates that hydrogen-driven microbial metabolisms prevail under the highly reducing, high pH conditions that characterize these environments. Laboratory experiments were carried out to optimize the growth conditions of sulfate reducing bacteria as possible analogs to populations that exist in the subseafloor near the Lost City, on the Atlantis Massif. These experiments were used to refine the analytical protocol used to establish more than 200 microcosm experiments on core material from the Atlantis Massif during IODP Expedition 357. Although in situ cell abundances in the core material were extremely low, respiratory activity coupled to sulfate reduction was detected in several instances, particularly when acetate was used as a growth substrate. Additional cultures of methanogens were used to investigate how the production and isotopic composition of carbon changes under different geochemical conditions. A change in the relative abundance of organic molecules was observed between the treatments. Similar processes occurring in the subseafloor may influence community structure as these products would support different types of secondary consumers. Carbon isotope signatures of biosynthetic products also changed substantially between the treatments. These results will help in future interpretations of biomarker signatures isolated from Atlantis Massif cores and Lost City chimneys.