We sampled fishes and aquatic insects monthly (Jun.–Sept. 2002) from intermittent tributaries of the Alum Fork of the Saline River (Arkansas, U.S.A.) to quantify the response of fish and aquatic insect assemblage properties to seasonal desiccation and habitat fragmentation. We collected a total of 4219 individuals, representing 18 species of fishes and 27 families of aquatic insects. Changes in the composition of fish assemblages were significantly related to temporal variability in pool volume and location in the watershed. Smaller, upstream pools varied in volume more than downstream pools. Fish assemblages were significantly more similar through summer in downstream, larger pool habitats. Changes in the composition of aquatic insect families were related to variation in water quality conditions. Highly eutrophied sites were typically dominated by midge larvae (Chironomidae), resulting in highly similar assemblages over time. Our results support findings at larger space and time scales and call attention to the importance of rapid changes in habitat quality, size and connectivity on stream communities.