The phylum Aquificae comprises chemolithoautotrophic thermophilic to hyperthermophilic bacteria, in which the nitrogenase reductase gene (nifH) has been reported. However, nitrogen-fixing activity has not yet been demonstrated in members of this deeply branching bacterial phylum. We isolated two thermophilic diazotrophic strains from chemosynthetic microbial communities in slightly alkaline hot springs (≥70°C) in Nakabusa, Nagano Prefecture, Japan. A phylogenetic analysis based on 16S rRNA genes identified these strains as members of the genus Hydrogenobacter within Aquificae. Their NifH sequences showed 96.5 and 97.4% amino acid sequence identities to that from Hydrogenobacter thermophilus TK-6. Nitrogenase activity, measured by acetylene reduction, was confirmed in both strains at 70°C. These novel strains grew under semi-aerobic conditions by using CO2 as the sole carbon source and N2 as the sole nitrogen source in media containing hydrogen and/or thiosulfate. To the best of our knowledge, this is the first demonstration of active nitrogen fixation in thermophilic bacteria at 70°C and in the phylum Aquificae.