Groundwater ecosystems have a diverse and unique fauna, often dominated by Crustacea and generally characterised by short range endemics confined to single aquifers. Much of this knowledge has come from studies conducted either in fractured rock aquifers or alluvial aquifers. Karstic subterranean environments are present in the Cambrian Limestone Aquifer (CLA) in the Northern Territory, Australia, a freshwater aquifer which spans an area of ~28,000 km2. The presence of underground caverns and channels potentially allows extensive connectivity within this groundwater system. The emerging shale gas industry in the Beetaloo region, which underlies the CLA, provided the impetus to undertake the first survey of the potential existence of a stygofaunal community. Twenty-six groundwater wells (bores) and two springs were sampled in August and October 2019, across a distance of ~500 km, from the sub-tropical Mataranka region in the north to the semi-arid Barkly Tablelands in the south. Plankton nets and motorised pumps were used to collect water samples and conventional microscope-based morphological examinations in conjunction with environmental DNA (eDNA) were used to determine the presence of stygofauna. COI barcoding and 16S rRNA regions were also used for phylogenetic analysis. All stygofaunal communities were dominated by crustaceans, namely shrimps, amphipods, ostracods, copepods and syncarids. This fauna showed little affinity with the stygofauna recorded from more extensively sampled aquifers in north-western Australia, with new genera and species present in the CLA. eDNA analysis showed the presence of diverse biota at sites where direct water sampling for intact animals was difficult. COI and 16S analysis confirmed that a species of blind shrimp, Parisia unguis, occurred extensively throughout the aquifer, over a distance of at least ~300 km. The presence of Pa. unguis at widely separated sites across the CLA is consistent with substantial connectivity within the aquifer. This connectivity indicates that the risk of groundwater contamination from fracking chemicals needs to be adequately mitigated to prevent widespread effects.