Nitrogen (N) bioavailability is a primary limiting nutrient for crop and feedstock productivity. Associative nitrogen fixation (ANF) by diazotrophic bacteria in root-zone soil microbial communities have been shown to provide significant amounts of N to some tropical grasses, but this potential in switchgrass, a warm-season, temperate, US native, perennial tallgrass has not been widely studied. ‘Alamo’ and ‘Dacotah’ are cultivars of switchgrass, adapted to the southern and northern regions of the United States, respectively, and offer an opportunity to better describe this plant–bacterial association. The nitrogenase enzyme activity, microbial communities, and amino acid profiles in the root-zones of the two ecotypes were studied at three different plant growth stages. Differences in the nitrogenase enzyme activity and free soluble amino acid profiles indicated the potential for greater nitrogen fixation in the high productivity Alamo compared with the lower productivity Dacotah. Changes in the amino acid profiles and microbial community structure (rRNA genes) of the root-zone suggest different plant–bacterial interactions can help to explain differences in nitrogenase activity. PICRUSt analysis revealed functional differences, especially nitrogen metabolism, that supported ecotype differences in root-zone nitrogenase enzyme activity. It is thought that the greater productivity of Alamo increased the belowground flow of carbon into roots and root-zone habitats, which in turn support the high energy demands needed to support nitrogen fixation. Further research is thus needed to understand plant ecotype and cultivar trait differences that can be used to breed or genetically modify crop plants to support root-zone associations with diazotrophs.