Extensive lacustrine microbialite deposits exposed along the shores of Great Salt Lake (GSL), Utah preserve a rich continental paleoenvironmental record. Newly‐reported microbialite carbon and oxygen stable isotope ratios in carbonate, nitrogen isotope ratios in organic matter, and organic matter radiocarbon ages archive paleolake hydrological and biogeochemical changes from the late Pleistocene through the Holocene. Positive correlations between δ18O and δ13C in ∼15 – 7.6 cal ka microbialite carbonate are consistent with a hydrologically closed‐basin lake with fluctuations in volume, chemistry, and associated changes in lake primary production. The δ15N of microbialite bulk organic matter (5 – 18 ‰ versus AIR) shows that the balance between nitrogen fixation and assimilation of dissolved inorganic nitrogen has varied significantly. Inverse δ18O and δ13C correlations in combination with high δ15N in some carbonate deposits may imply periods of higher salinity and stable lake stratification similar to modern GSL conditions. We compare our C and O data sets with Pleistocene Lake Bonneville carbonate stable isotope records and demonstrate progressive development of spatially‐isolated hydrological basins during the shift to warmer and drier conditions in the Holocene.