The 715-km Natchez Trace Parkway (National Park Service) passes through portions of three ecological regions (Mississippi Valley Loess Plains, Southeastern Plains and Interior Plateau) from its origin at Natchez, MS to its terminus near Nashville, TN. The park presents a latitudinal gradient with streams that are easily accessible within the park boundary and that exhibit a corresponding variety in physicochemical features. We examined 14 stream systems along this gradient and evaluated microbial community composition across separate stream sites, comparing stream water and leaf litter communities, and among leaf types by examining carbon utilization patterns (Biolog EcoPlates). Results from principal components analysis suggest that there were bigger differences in microbial community composition associated with leaf litter than with stream water. Within ecoregions, streams grouped differently when using water or leaf communities. Overall, communities present on sweetgum and oak litter could be separated from communities on tulip poplar and sycamore litter. These patterns suggest that differences in conditions at smaller scales, stream or litter, are more important than ecoregion differences in determining microbial community composition.