Aerobic soil bacteria metabolize atmospheric hydrogen to provide energy in nutrient poor environments. This process is mediated by [NiFe]-hydrogenases capable of oxidising H2 at atmospheric concentration. The soil saprophyte Mycobacterium smegmatis has two such phylogenetically distinct [NiFe]-hydrogenases, designated Huc and Hhy. Both hydrogenases are oxygen-tolerant, oxidise H2 to subatmospheric concentrations, and enhance survival during hypoxia and carbon limitation. Why then does M. smegmatis require two hydrogenases mediating a seemingly similar function? We resolve this by showing that Huc and Hhy are differentially expressed, localised, and integrated into the respiratory chain. Huc is active in late exponential and early stationary phase, supporting energy conservation during mixotrophic growth and the transition into dormancy. In contrast, Hhy is most active during persistence, providing energy for maintenance processes when carbon sources are depleted. Both hydrogenases are obligately linked to the respiratory chain via the menaquinone pool, but differentially interact with the terminal oxidases. Huc exclusively donates electrons to, and possibly physically associates with, the proton-pumping cytochrome bcc-aa3 supercomplex. In contrast, the more promiscuous Hhy can also provide electrons to cytochrome bd oxidase. These data demonstrate that, despite their similarities, Huc and Hhy perform distinct functions during mycobacterial growth and non-replicative persistence.