Laboratory studies have documented the extensive architectural differentiation of biofilms into complex structures, including filamentous streamers generated by turbulent flow. Still, it remains elusive whether this spatial organization of natural biofilms is reflected in the community structure. We analyzed bacterial community differentiation between the base and streamers (filamentous structures floating in the water) of stream biofilms under various flow conditions using denaturing gradient gel electrophoresis (DGGE) and sequencing. Fourth-corner analysis showed pronounced deviation from random community structure suggesting that streamers constitute a more competitive zone within the biofilm than its base. The same analysis also showed members of the a-Proteobacteria and Gemmatimonadetes to preferentially colonize the biofilm base, whereas b-Proteobacteria and Bacteroidetes were comparatively strong competitors in the streamers. We suggest this micro-scale differentiation as a response to the environmental dynamics in natural ecosystems.