Changes in surface land use may threaten groundwater quality and ecosystem integrity, particularly in shallow aquifers where links between groundwater and surface activities are most intimate. In this study we examine the response of groundwater ecosystem to agricultural land uses in the shallow alluvial aquifer of the Gwydir River valley, New South Wales, Australia. We compared groundwater quality and microbial and stygofauna assemblages among sites under irrigated cropping, non-irrigated cropping and grazing land uses. Stygofauna abundance and richness was greatest at irrigated sites, with the composition of the assemblage suggestive of disturbance. Microbial assemblages and water quality also varied with land use. Our study demonstrates significant differences in the composition of groundwater ecosystems in areas with different surface land use, and highlights the utility of groundwater biota for biomonitoring, particularly in agricultural landscapes.