Although it has been more than 30 years since the discovery of deep-sea hydrothermal vents, comprehending the interconnections between hydrothermal venting and microbial life remains a challenge. Here we investigate abiotic–biotic linkages in low-temperature hydrothermal biotopes at Desperate and Lilliput on the southern Mid-Atlantic Ridge. Both sites are basalt-hosted and fluids exhibit the expected chemical signatures. However, contrasting crustal permeabilities have been proposed, supporting pervasive mixing at Desperate but restricting circulation at Lilliput. In Desperate fluids, sulfide and O2 were readily available but H2 hardly detectable. Under incubation conditions (oxic unamended, sulfide-spiked, oxic and anoxic H2-spiked at 18°C), only sulfide oxidation byThiomicrospirafuelled biomass synthesis. Microbial phylogenies from Desperate incubation experiments resembled those of the natural samples suggesting that the incubation conditions mimicked the environment. In Lilliput fluids, O2 was limited, whereas sulfide and H2 were enriched. Autotrophy appeared to be stimulated by residual sulfide and by amended H2. Yet, based on bacterial phylogenies only conditions in anoxic H2-spiked Lilliput incubations appeared similar to parts of the Lilliput habitat. In anoxic H2-spiked Lilliput enrichmentsCampylobacteraceaelikely supported biomass production through H2 oxidation. We argue that the diverging circulation patterns arising from different subseafloor permeabilities act as major driving forces shaping these biotope structures.