Type 2 NADH dehydrogenase (Ndh-2) is an oxidative phosphorylation enzyme discussed as a promising drug target in different pathogens, including Plasmodium falciparum and Mycobacterium tuberculosis (Mtb). To kill Mtb, Ndh-2 needs to be inactivated together with the alternative enzyme type 1 NADH dehydrogenase (Ndh-1), but the mechanism of this synthetic lethality remained unknown. Here, we provide insights into the biology of NADH dehydrogenases and a mechanistic explanation for Ndh-1 and Ndh-2 synthetic lethality in Mtb. NADH dehydrogenases have two main functions: maintaining an appropriate NADH/NAD+ ratio by converting NADH into NAD+ and providing electrons to the respiratory chain. Heterologous expression of a water forming NADH oxidase (Nox), which catalyzes the oxidation of NADH, allows to distinguish between these two functions and show that Nox rescues Mtb from Ndh-1/Ndh-2 synthetic lethality, indicating that NADH oxidation is the essential function of NADH dehydrogenases for Mtb viability. Quantification of intracellular levels of NADH, NAD, ATP, and oxygen consumption revealed that preventing NADH oxidation by Ndh-2 depletes NAD(H) and inhibits respiration. Finally, we show that Ndh-1/ Ndh-2 synthetic lethality can be achieved through chemical inhibition.