Physical space is a fundamental habitat constraint for interstitial space-dwelling organisms; however, few studies have examined how physical space variation structures predator/prey interactions within such communities. Streambeds in the western Ozark Plateau are composed of Silurian/Ordovician chert gravel and contain a rich assemblage of interstitial space-dwelling species, including macroinvertebrates, fishes and aquatic salamanders. Here, we used field and lab experiments to examine interstitial space size preference and structure of an Ozark streambed community, with emphasis on the Oklahoma salamander, Eurycea tynerensis, and its predators (crayfish) and prey (isopods and amphipods). In the field, we implemented a three by six grid of enclosures containing three distinct size classes of well-sorted substrate (small, medium and large) that represented three categories of interstitial space size. Isopods and amphipods were the most abundant macroinvertebrates at the site, and the average size of isopods increased with substrate size. Salamanders were significantly more abundant in medium-sized substrate enclosures, whereas isopods of consumable size and amphipods were more abundant in small and medium substrate, and crayfish were found exclusively in large substrate enclosures. Pairwise choice experiments in the lab showed that salamanders always preferred the largest gravel size (i.e. large or medium to small, and large to medium). A subsequent experiment performed with uniformly large gravel demonstrated that salamanders avoided positions near and adjacent to crayfish. We suggest that the finite interstitial space distribution of Oklahoma salamanders is limited by physical constraints of small, prey-rich spaces, and avoidance of predators and prey scarcity in the interstitial spaces among large substrates. This study demonstrates the strong influence of interstitial space size on community structure and predator/prey interactions in chert-bottomed Ozark streams.