Culverts can provide a significant barrier to fish passage by fragmenting fish habitats and impeding the passage success of small-bodied fish. Geographical connectivity is critical to the maintenance of diverse fish assemblages. Culverts with high cross-sectional velocity can cause population fragmentation by impeding passage of small, freshwater fish. Behavioral responses of small fish to high velocities can differ among functional groups, and swimming behavior of many species is not well known. We tested effects of substrate type on swimming behavior in two small, freshwater fish species—southern leatherside chub (Lepidomeda aliciae, a midwater species), and longnose dace (Rhinichthys cataractae, a benthic species)—across three substrate treatments: (1) a bare flume, (2) large flow obstacles, and (3) a natural cobble substrate. Both longnose dace and southern leatherside chub used paths of low velocity and swam in the near-substrate boundary area. Fish in the bare flume and large obstacle treatments swam along the corners of the flume in a straight swim path, whereas fish in the natural substrate treatment used all parts of the flume bed. There was no relationship between passage success of fish and substrate type, fish species, or their interaction. In contrast, substrate type, fish species, and their interaction were significant predictors of passage time. Southern leatherside chub passed through the test section about two to four times faster than longnose dace. Both species took longer to pass through the large flow obstacle treatment compared to the bare flume or natural substrate. The natural substrate created a complex velocity profile with areas of low velocity throughout the entire flume, in contrast to the other two treatments. Our data suggest natural substrates can improve the passage of small fish in high-velocity culverts for both benthic and midwater functional groups.