Groundwater ecosystems are globally widespread yet still poorly understood. We investigated the microbiology of >100 groundwater samples from 90 monitoring wells (<250m depth) located in 14 aquifers in the Canadian Prairie. This dataset contains the nucleotide sequences and taxonomy of bacterial 16S rRNA gene amplicons retrieved from groundwater samples of 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 DNA extraction, sequencing and sequence analyses can be found in the associated publication by Ruff et al. 2023.