Serpentinization is the hydration and oxidation of ultramafic rock, which occurs as oceanic lithosphere is emplaced onto continental margins (ophiolites), and along the seafloor as faulting exposes this mantle-derived material to circulating hydrothermal fluids. This process leads to distinctive fluid chemistries as molecular hydrogen (H2 ) and hydroxyl ions (OH(-) ) are produced and reduced carbon compounds are mobilized. Serpentinizing ophiolites also serve as a vector to transport sulfur compounds from the seafloor onto the continents. We investigated hyperalkaline, sulfur-rich, brackish groundwater in a serpentinizing continental ophiolite to elucidate the role of sulfur compounds in fuelling in situ microbial activities. Here we illustrate that key sulfur-cycling taxa, including Dethiobacter, Desulfitispora and ‘Desulforudis’, persist throughout this extreme environment. Biologically catalysed redox reactions involving sulfate, sulfide and intermediate sulfur compounds are thermodynamically favourable in the groundwater, which indicates they may be vital to sustaining life in these characteristically oxidant- and energy-limited systems. Furthermore, metagenomic and metatranscriptomic analyses reveal a complex network involving sulfate reduction, sulfide oxidation and thiosulfate reactions. Our findings highlight the importance of the complete inorganic sulfur cycle in serpentinizing fluids and suggest sulfur biogeochemistry provides a key link between terrestrial serpentinizing ecosystems and their submarine heritage.