Understanding environmental factors that influence obligate groundwater dwelling (stygobiotic) fauna is crucial for groundwater ecosystem monitoring and management. Field studies have indicated geological factors are a major influence on the abundance and richness of stygofauna, however the precise mechanisms and true influence of the aquifer sediment matrix on biota is unclear. In this study we examined the habitat use and preferences, in terms of sediment particle sizes, of stygobiotic meiofauna (Harpacticoida and Cyclopoida Copepoda), and macroinvertebrates (Amphipoda and Syncarida) using laboratory microcosms. We first tested the ability of each taxon to use (move into) clay (< 0.06 mm), sand (0.3–0.7 mm) and gravel sediments (2–4 mm). Subsequently, the preference for each sediment was compared by examining the distribution of animals in microcosms containing two different sediment types. Both the harpacticoids and cyclopoids were able to use clay, whereas larger amphipods and syncarids mostly remained on the sediment surface. All taxa were able to use sand and gravel substrates. Amphipods preferred gravel over sand and clay. Both copepods and syncarids preferred sand and gravel over clay, but showed no preference between gravel and sand. This study demonstrates the general inability of some stygobiotic macroinvertebrates to use clay sediments and overall differences in sediment use among stygobiotic meio- and macrofauna. From these findings, the typically heterogenous distributions and diversity of stygofauna observed in field studies may be related to variability in sediment composition.