Rising demands for both food and energy have lifted up the idea of producing renewable biofuels from bioenergy grasses that can companion with associative N2ixing bacteria. Associative N2ixing bacteria can partially fulfill the N requirements of bioenergy grasses enabling successful application for marginal lands. The overall objective of this study was to measure the potential N2ixation of bacteria associated with three bioenergy grasses, giant miscanthus (Miscanthus x giganteus), switchgrass (Panicum virgatum; ‘Alamo’) and energycane (hybrid of Saccharum spontaneum and S. officinarum; HO 02-147) as well as identifying N2ixing bacteria associated with these energy grasses and applying these isolates to grasses to increase biomass yield. It was hypothesized that three different energy grasses, giant miscanthus, switchgrass, and energycane have different capabilities to support the growth of associative N2ixing bacteria, and thus exhibit different rate and quantities of N2ixation. The dynamics of delta15Nair in the root-zones of three perennial bioenergy grasses, biomass yield and nitrogen derived from (%Ndfa) were obtained. Greater potential for N2ixation was identified in energycane associated plant system compared to switchgrass and giant miscanthus. Sorghum bicolor (M81-E) was used to calculate the %Ndfa. Moreover, a 15N2 enriched greenhouse study was carried out to estimate and compare the contribution of ANF to support field data. Greenhouse data further confirmed that energycane associated bacteria has greater potential N2ixation compared to giant miscanthus and switchgrass. Composition and diversity of N2ixing bacteria associated as endophytes and in the rhizosphere community of these three energy grasses were identified in marginal lands. Diverse N2ixing bacteria were observed to be associated with different grasses and Azospirillum sp. was identified in energycane. The effect of N2ixing bacteria on biomass was studied by inoculating three energy grasses with the bacteria isolated from energycane. Increased root lengths of giant miscanthus were observed upon inoculation. However, no other changes in biomass yield or shoot lengths were observed in three bioenergy grasses. Further research is needed to quantify endophytic and rhizosphere N2ixing bacteria and to determine their role in association in an effort to increase the biomass yield of other grasses and reduce N fertilizer inputs.