Water chemistry, energetic modeling, and molecular analyses were combined to investigate the microbial ecology of a biofilm growing in a thermal artesian spring within Hot Springs National Park, AR. This unique fresh water spring has a low dissolved chemical load and is isolated from both light and direct terrestrial carbon input – resulting in an oligotrophic ecosystem limited for fixed carbon and electron donors. Evaluation of energy yields of lithotrophic reactions putatively linked to autotrophy identified the aerobic oxidation of methane, hydrogen, sulfide, ammonia, and nitrite as the most exergonic. Small subunit (SSU) rRNA gene libraries from biofilm revealed a low-diversity microbial assemblage populated by bacteria and archaea at a gene copy ratio of 45:1. Members of the bacterial family ‘Nitrospiraceae’, known for their autotrophic nitrite oxidation, dominated the bacterial SSU rRNA gene library (approximately 45%). Members of the Thaumarchaeota ThAOA/HWCGIII (>96%) and Thaumarchaeota Group I.1b (2.5%), which both contain confirmed autotrophic ammonia oxidizers, dominated the archaeal SSU rRNA library. Archaea appear to dominate among the ammonia oxidizers, as only ammonia monooxygenase subunit A (amoA) genes belonging to members of the Thaumarchaeota were detected. The geochemical, phylogenetic, and genetic data support a model that describes a novel thermophilic biofilm built largely by an autotrophic nitrifying microbial assemblage. This is also the first observation of ‘Nitrospiraceae’ as the dominant organisms within a geothermal environment.