The lipid-rich cell wall of mycobacteria is essential not only for virulence but also for survival. Whilst anabolic pathways for mycobacterial lipid biosynthesis have been well studied, there has been little research looking into lipid catabolism. The genome of Mycobacterium tuberculosis encodes multiple enzymes with putative roles in the β-oxidation of fatty acids. In this report we explore the functionality of FadB2, one of five M. tuberculosis homologues of a β-hydroxybutyryl-CoA dehydrogenase, an enzyme that catalyses the third step in the β-oxidation cycle. Purified M. tuberculosis FadB2 catalysed the in vitro NAD+-dependent dehydration of β-hydroxybutyryl-CoA to acetoacetyl-CoA at pH 10. Mutation of the active-site serine-122 residue resulted in loss of enzyme activity, consistent with the function of FadB2 as a fatty acyl dehydrogenase involved in the β-oxidation of fatty acids. Surprisingly, purified FadB2 also catalysed the reverse reaction, converting acetoacetyl-CoA to β-hydroxybutyryl-CoA, albeit in a lower pH range of 5.5–6.5. Additionally, a null mutant of fadB2 was generated in Mycobacterium smegmatis. However, the mutant showed no significant differences from the wild-type strain with regard to lipid composition, utilization of different fatty acid carbon sources and tolerance to various stresses; the absence of any phenotype in the mutant strain could be due to the potential redundancy between the five M. smegmatis fadB paralogues.