Nitrososphaera viennensis is a newly isolated ammonia-oxidizing archaeon from soil, belonging to the Thaumarchaeota group 1.1b. This study describes the cultivation and isolation of this archaeon, which grows on ammonia or urea as an energy source and can tolerate higher ammonia concentrations than the marine isolate Nitrosopumilus maritimus. Unlike N. maritimus, which is strictly chemolithoautotrophic, N. viennensis requires addition of low amounts of pyruvate or coculture with bacteria to achieve high growth rates. The findings expand the known metabolic capabilities of AOA and help explain previous controversies regarding their activity and carbon assimilation. The archaeon was isolated from garden soil in Vienna and grown in enrichment cultures with mineral media. Two stable enrichments, EN76 and EN123, were obtained, with EN76 showing the highest archaeal abundance. The genome of EN76 was sequenced and revealed genes for urease, indicating the ability to use urea as a nitrogen source. The optimal growth temperature for EN76 was slightly above 35°C, and the optimal pH was slightly above 7.5. The pure culture of EN76 grew well on ammonium and supplemented with pyruvate, but growth was significantly slower under purely autotrophic conditions. NanoSIMS analysis showed that carbon from pyruvate was incorporated into the biomass of EN76, indicating mixotrophic growth. However, less than 10% of the cellular carbon came from pyruvate, suggesting that carbon assimilation was mainly driven by bicarbonate fixation. The study also revealed that N. viennensis is closely related to Candidatus N. gargensis, an AOA that has not yet been obtained in pure culture. The isolation of N. viennensis provides new insights into the energy metabolism and carbon assimilation of AOA, which are among the most abundant but poorly understood organisms on Earth. The study highlights the importance of obtaining pure cultures to better understand the growth requirements of AOA. The new archaeon is proposed as a new species, Nitrososphaera viennensis, belonging to the newly proposed order Nitrososphaerales, family Nitrososphaeraceae.Nitrososphaera viennensis is a newly isolated ammonia-oxidizing archaeon from soil, belonging to the Thaumarchaeota group 1.1b. This study describes the cultivation and isolation of this archaeon, which grows on ammonia or urea as an energy source and can tolerate higher ammonia concentrations than the marine isolate Nitrosopumilus maritimus. Unlike N. maritimus, which is strictly chemolithoautotrophic, N. viennensis requires addition of low amounts of pyruvate or coculture with bacteria to achieve high growth rates. The findings expand the known metabolic capabilities of AOA and help explain previous controversies regarding their activity and carbon assimilation. The archaeon was isolated from garden soil in Vienna and grown in enrichment cultures with mineral media. Two stable enrichments, EN76 and EN123, were obtained, with EN76 showing the highest archaeal abundance. The genome of EN76 was sequenced and revealed genes for urease, indicating the ability to use urea as a nitrogen source. The optimal growth temperature for EN76 was slightly above 35°C, and the optimal pH was slightly above 7.5. The pure culture of EN76 grew well on ammonium and supplemented with pyruvate, but growth was significantly slower under purely autotrophic conditions. NanoSIMS analysis showed that carbon from pyruvate was incorporated into the biomass of EN76, indicating mixotrophic growth. However, less than 10% of the cellular carbon came from pyruvate, suggesting that carbon assimilation was mainly driven by bicarbonate fixation. The study also revealed that N. viennensis is closely related to Candidatus N. gargensis, an AOA that has not yet been obtained in pure culture. The isolation of N. viennensis provides new insights into the energy metabolism and carbon assimilation of AOA, which are among the most abundant but poorly understood organisms on Earth. The study highlights the importance of obtaining pure cultures to better understand the growth requirements of AOA. The new archaeon is proposed as a new species, Nitrososphaera viennensis, belonging to the newly proposed order Nitrososphaerales, family Nitrososphaeraceae.