One of the most challenging questions in the assessment of groundwater ecosystem health is whether assemblages of groundwater biota can be used reliably as biomarkers. In this study, we aimed to (a) explore the diversity, distribution, and ecological composition of groundwater Crustacea, Copepoda, and Ostracoda communities from six aquifers in Central Spain and (b) relate the structure of the species assemblages to the aquifers' hydrogeological conditions, habitat features, and hydrochemistry. The datasets were simultaneously collected from 24 boreholes drilled in six unconfined aquifers from the upper part of the Tajo catchment. Our results show that the assemblages are primarily influenced by the specific hydrogeological conditions of the aquifer, determined by groundwater flow, supply of organic matter from the surface, and habitat features, whereas the aquifer's hydrochemistry has only a marginal effect. The assemblages from the Tertiary detrital aquifer were the most diverse, containing a mixture of stygobiont and nonstygobiont species distributed preferentially in the recharge area of the aquifer, characterized by medium hydraulic conductivity, high surface input and relatively high water renewal. The Quaternary alluvial aquifer type was found to have a moderate richness with several hyporheic dwellers, indicating an active surface/groundwater exchange with the adjacent stream channel.In contrast, the carbonate aquifer type was less diverse but markedly distinct owing to the high occurrence of stygobionts. Our results provide a step forward in exploring the groundwater biodiversity of aquifers in Spain and towards incorporating an ecological perspective into overall assessments of groundwater status.