Groundwater is a critical freshwater resource across northern Australia, yet the microbiomes of its aquifers are almost entirely uncharacterised. This knowledge gap is critical: new land uses, including onshore shale gas extraction in the Beetaloo Sub-basin and intensive irrigated agriculture, are expanding across regions that overlie major aquifers. Groundwater microbiomes (dominated by prokaryotes and microeukaryotes) are difficult to characterise using the morphological approaches typically applied in biomonitoring. Here we use eDNA metabarcoding to provide the first multi-domain (prokaryotes, fungi, amoebae and ciliates) description of groundwater microbiomes across a ~1,000 km tropical-to-arid climatic gradient in the Northern Territory, Australia. A citizen science sampling protocol, supported by a smartphone app and project website, enabled collection of samples from 41 bores spanning six regions overlying major aquifers. Samples of potable (treated) water from four NT towns were included as a biologically-depleted reference to benchmark assay performance and provide context for non-scientists. All four biotic groups exhibited significant but partial regional structuring and consistently high taxonomic turnover among bores, with no single ASV contributing more than ~0.3–11% of between-region dissimilarity. After accounting for broad-scale geography, EC and pH explained only a small fraction of community composition across all groups. These patterns indicate that no single bore is representative of the broader regional microbiome, with direct implications for the design of groundwater biomonitoring programs. The multi-domain microbiome baselines established here are an essential pre-impact reference against which future changes associated with expanding resource development and land-use change can be detected and assessed.