In semi-arid regions, such as Australia’s Northern Territory wet-dry tropics, groundwater represents a critical freshwater resource supporting both anthropogenic needs and unique subterranean ecosystems. The Beetaloo Sub-basin, a 30,000 km2 area highly prospective for shale gas extraction, overlies complex aquifer systems that harbor stygofauna—obligate groundwater-dwelling invertebrates—and diverse microbial communities. An improved understanding of these subterranean ecosystems is necessary for assessing the ecological risks associated with onshore petroleum activities. Here, we investigated the distribution and ecology of stygofauna from three seasonal surveys conducted between October 2022 and May 2024. A multi-methodological approach was employed, integrating physical net hauling, video borescope inspections, and environmental DNA (eDNA) metabarcoding (COI and 18S rRNA) to characterise water quality, microbial community structure, and faunal diversity. Stygofauna were detected in 15 of 33 successfully surveyed bores, primarily within the Tindall Limestone aquifer. Taxonomic analysis identified seven distinct groups, dominated by crustaceans, including genetically distinct populations of the decapodan shrimp Parisia unguis. While water chemistry and depth (9–100 m) were poor predictors of stygofaunal presence, video evidence confirmed that shrimp were strongly associated with specific geological cavities and bore screenings. Metagenomic analyses revealed a complex microbial landscape influenced by bore architecture and geography. In some subterranean ecosystems terrestrial tree roots can act as the primary source of carbon for food webs. However, in the Beetaloo we observed stygofauna up to 100m below the surface in waters depleted in dissolved organic carbon (<1mg L-1) coupled with the presence of diverse chemolithotrophic microorganisms. These results suggest that the oxidation of inorganic molecules by chemolithotrophs may serve as the primary energy pathway for invertebrates, forming the foundation of the subterranean food web. We demonstrate that no single sampling method ensures total detection of stygofauna, highlighting the necessity of integrated monitoring approaches. The discovery of localised, genetically distinct populations suggests limited connectivity in some areas, increasing vulnerability to environmental disturbances. Our research provides a baseline for environmental impact assessments, underscoring that alterations to nutrient profiles or microbial structures could destabilise these cryptic ecosystems.