The deep continental crust represents a vast potential habitat for microbial life where its activity remains poorly constrained. A common characteristic of these ecosystems is the presence of organic acids like acetate, but the role of these molecules in the subsurface carbon cycle - including the mechanism and rate of their turnover - is still unclear. Here, we developed an isotope-exchange ‘clock’ based on the temperature-dependent abiotic equilibration of H-isotopes between acetate’s methyl-group and water, which can be used to define the maximum in situ residence time for acetate. We applied this technique to the fracture fluids in Birchtree and Kidd Creek mines within the Canadian Precambrian crust. At both sites, we found isotopic disequilibrium between acetate and water, indicating acetate residence times <1 million years and a rate of turnover that could theoretically support microbial life. However, radiolytic water-rock reactions could also contribute to acetate production and degradation, a process that would have global relevance for the deep biosphere. More broadly, our study demonstrates that isotope-exchange clocks can constrain in situ residence times of biomolecules with possible applications to other environments.