Groundwater ecosystems are globally widespread yet still poorly understood. This dataset contains values and calculations to estimate microbial oxygen consumption in groundwater samples from aquifers in the Canadian Prairie, collected between 2015 and 2020. The study area comprised 14 major aquifers and a geographic area of ~210.000 km2. The goal of the study was to understand the links between the biogeochemistry and microbial ecology of groundwater ecosystems in diverse geological settings on a broad spatial scale. Details concerning microbial cell counts and oxygen consumption can be found in the associated publication by Ruff et al. 2023.