Short-range endemism is common in groundwater fauna (stygofauna), placing many species at risk from anthropogenic impacts such as water abstraction and pollution. Few of the alluvial aquifers in eastern Australia have been sampled for stygofauna. Fauna from two aquifers in Queensland and two in New South Wales was sampled to improve ecological knowledge of stygofauna and the potential threats posed to it by development. Our surveys found stygofauna in all four aquifers, with most taxa collected from bores with low electrical conductivity (<1500 μS cm⁻¹). Taxon richness decreased with distance below the water table. The most taxon-rich bores in each region occurred where the water table depth was <10 m, were associated with the alluvium of tributaries of large regulated river systems, and were near phreatophytic trees. It is possible that tree roots constitute a habitat and source of organic matter in alluvial aquifers as they do in cave streams. It is important to document the biodiversity of particular regions and aquifers so that species can be conserved in the face of increasing groundwater use. For effective resource management, future research should strive to understand the tolerances and ecological requirements of groundwater communities and the ecosystem services they provide.