Groundwater is essential to crop production in many parts of the world, and the provision of clean groundwater is dependent on healthy groundwater ecosystems. To understand better the functioning of groundwater ecosystems, it is necessary to understand how the biota responds to environmental factors, and so distinguish natural variation from human induced changes. This study compares the groundwater biota of the adjacent Gwydir and Namoi River alluvial aquifers, both in the heartland of Australia's cotton industry, and investigates the relative importance of environmental, anthropogenic, geological, and evolutionary processes on biotic distribution. Distinct differences in biotic assemblages were recorded between catchments at a community level. However, at a functional level (e.g. microbial activity, stygofauna abundances and richness) both ecosystems were similar. The distribution of biota in both catchments was influenced by similar environmental variables (e.g. geology, carbon availability, season, and land use). Broad trends in biotic distribution were evident: stygofauna responded most strongly to geological variables (reflecting habitat) and microbes to water quality and flow. Agricultural activities influenced biota in both catchments. Although possessing different taxa, the groundwater ecosystems of the two aquifers were functionally similar and responded to similar environmental conditions.