Soils and waters were collected from Great Smoky Mountains National Park (GSMNP), and bacteria were cultured or DNA cloned from the samples. Oconaluftee Visitors Center and Kephart Prong Trail were sampled to examine distributions of heterotrophs inhabiting streams and riparian soil. Soil from All Taxa Biodiversity Inventory plots at Albright Grove, Cataloochee, and Purchase Knob were also sampled. A total of eleven phyla were detected, of which six were only found via culture-independent techniques. Overall, 69 genera were documented, with differences in their detection in soil and water and by methodology. Firmicutes dominated cultures from soil, while Acidobacteria dominated clone libraries; Bacteroidetes was the dominant phylum in water. Three classes of the phylum Proteobacteria were commonly seen as isolates or clones. Prokaryotic diversity is extraordinary; this is the first inventory examining non-photosynthetic bacteria inhabiting GSMNP and it lays the groundwork for investigations exploring the true breadth of diversity in the park and what this diversity means to the broader ecosystem.