Around 50% of humankind relies on groundwater aquifers as a source of drinking water. We investigated the age, geochemistry, and microbiology of 138 groundwaters from 87 monitoring wells (<250m depth) located in 14 aquifers in the Canadian Prairie. This data collection contains geological, physical, geochemical, gas, isotope and other contextual data, as well as microbial cell counts, 16S rRNA gene sequences, community diversity metrics and micrographs. Geochemistry and microbial ecology showed consistent trends suggesting large-scale aerobic and anaerobic hydrogen, methane, nitrogen, and sulfur cycling carried out by diverse microbial communities. Older groundwaters, especially of organic carbon-rich strata, contained on average more cells (up to 1.4'107/mL) than younger groundwaters, challenging current estimates of global groundwater population sizes. Substantial concentrations of dissolved oxygen (0.52±0.12mg/L [mean±SE]; n=57) in older groundwaters could support aerobic lifestyles in subsurface ecosystems at an unprecedented scale. Metagenomics, oxygen isotope analyses and mixing models indicated that “dark oxygen” was produced in situ via microbial dismutation. We show that ancient groundwaters sustain productive communities and highlight an overlooked oxygen source in present and past subsurface ecosystems of Earth.