Deep fracture-hosted fluids of Precambrian bedrock cratons are relatively stagnant over long time spans compared to near-surface systems. However, episodic events, such as fracture reactivations, transgressions, and deglaciations, may introduce dilute water, replacing, and mixing with the deep continental brines, thereby sparking microbial activity. Secondary minerals that line bedrock fractures serve as important geochemical archives for such episodic events. Here we explore the fracture mineral record of Archean rocks of Western Greenland by analyzing samples from deep boreholes with the aim to trace and characterize episodic paleofluid flow and paleomicrobial activity. A sequence of hydrothermal to low temperature fluid flow events is demonstrated. For the youngest generation, microscale S-isotope analysis of pyrite reveals substantial 34S-depletion (minimum δ34S:−58‰V-CDT) compared to fracture-hosted barite (δ34S:13‰ ± 2‰) and gypsum (δ34S:2.6‰–10.6‰). This suggests the formation of pyrite following S isotope fractionation during microbial sulfate reduction. This metabolism is further indicated by several methyl-branched fatty acids preserved in calcite. A general discrepancy between calcite and groundwater δ18O-values suggests that calcite formed from water different from the presently residing glacial meltwater-influenced groundwater mix. High spatial resolution U-Pb carbonate geochronology of the youngest generation of calcite yielded ages for two samples: 64 ± 3, 75 ± 7 Ma (2σ). These ages overlap with tectonic events related to early stages, or prestages, of the opening of the Atlantic and Labrador Seas. This suggests that deep fracture networks in Western Greenland were colonized by microorganisms, such as sulfate reducers, in the course of this extensional event.