The Na⁺‑translocating NADH:quinone oxidoreductase (Na⁺‑NQR) is a central enzyme in the respiratory chain of many Gram-negative bacteria including pathogens such as Vibrio cholerae. Oxidation of NADH and reduction of ubiquinone by Na⁺‑NQR is usually followed in the presence of oxygen, but this results in the formation of harmful reactive oxygen species and sub-stoichiometric reduction of ubiquinone. Therefore, activity measurements were performed under anaerobic conditions revealing high affinities of Na⁺ and ubiquinone towards the Na⁺-NQR from Vibrio cholerae.