Serpentinization – the hydrothermal alteration of ultramafic rocks – is a unique mineralogical process that results in H2 and CH4 – rich fluids that can support microbial communities. Using a combination of petrographic observations, bulk rock and in-situ sulfur isotope signatures of variably serpentinized peridotites from four different geotectonic environments we provide new constraints on the factors that support microbial activity. The studied samples overall suggest that redox conditions reflected by the presence of pyrite and pyrrhotite represent the fluid chemistry that favors microbial activity. This fluid chemistry, i.e., the prevailing hydrogen and sulfur fugacities of the fluid, are thereby correlated to high water-rock ratios and increased incorporation of seawater-derived species such as sulfate and carbonate. These species serve as major energy sources of microbial activity. In contrast, highly reducing conditions and limited fluid input limits or even prevents microbial activity within serpentinites due to insufficient availability of these species. Interaction with carbonate and sulfate-bearing meteoric waters is likely an essential process that supports microbial activity in continental serpentinization environments. Overall, this study shows that apart from fluid temperatures being within the limits of life, the fluid chemistry (redox conditions and availability of e.g. carbonate and sulfate) are the primary factors that control the presence or absence of microbial communities within serpentinizing peridotites. This study highlights the importance of combining bulk rock and in-situ stable isotope data with petrographic and mineralogical observations in order to better constraint the presence of microbial communities within the subsurface of peridotite-hosted hydrothermal systems.