Intermittent rivers and ephemeral streams are critical yet understudied ecosystems, particularly in semi-arid regions where hydrologic variability often governs community structure and ecosystem processes. This study examined how variation in hydrologic regimes affected the diversity and composition of fish and benthic invertebrate communities and food web characteristics. We examined these responses in nine study sites in the San Saba River (TX, USA) in which sites had one of three hydrologic regimes (perennial and temporally stable flows, intermittent flows, and seasonally variable but perennial flows) at two seasonal periods (late Spring; growing season versus early Fall; dry season). Riffle and pool habitats at each site were sampled twice and water chemistry, benthic algae, terrestrial detritus, macroinvertebrates, and fish were sampled in both mesohabitat types. Diversity (taxonomic richness) of macroinvertebrates and fish were assessed within and across mesohabitats, sites, as well as across hydrological regime types. Stable isotopes of C and N were used to examine food chain length, basal resource dependance (allochthonous versus autochthonous) among mesohabitats, sites, and hydrologic regimes. Invertebrate taxonomic richness was greater in hydrologically consistent sites when compared to sites with more variable discharge regimes, but riffles habitats consistently had greater invertebrate richness than pools. Similarly, fish taxonomic richness was greater in perennially flowing sites, but the greatest richness was in pool habitats. The trophic position of blacktail shiner (Cyprinella venusta), a cosmopolitan fish species found at all sites, was one trophic level lower at intermittent flow sites when compared to the perennially flowing sites, indicating that variability in discharge and the number of days with zero flow influences the length of food chains. Cumulatively, these results indicate that the effects of flow regime variability on richness and food web characteristics depend on the specific group of study organisms which are examined and the mesohabitat unit that is sampled.