Rifabutin is a spiro-piperidyl-rifamycin structurally closely related to rifampicin that shares many of its properties. We attempted to address the reasons why this drug, which was recently recognized as a WHO Essential Medicine, still had a far narrower range of indications than rifampicin, 24 years after its launch. In this comprehensive review of the classic and more recent rifabutin experimental and clinical studies, the current state of knowledge about rifabutin is depicted, relying on specific pharmacokinetics, pharmacodynamics, antimicrobial properties, resistance data and side effects compared with rifampicin. There are consistent in vitro data and clinical studies showing that rifabutin has at least equivalent activity/efficacy and acceptable tolerance compared with rifampicin in TB and non-tuberculous mycobacterial diseases. Clinical studies have emphasized the clinical benefits of low rifabutin liver induction in patients with AIDS under PIs, in solid organ transplant patients under immunosuppressive drugs or in patients presenting intolerable side effects related to rifampicin. The contribution of rifabutin for rifampicin-resistant, but rifabutin-susceptible, Mycobacterium tuberculosis isolates according to the present breakpoints has been challenged and is now controversial. Compared with rifampicin, rifabutin's lower AUC is balanced by higher intracellular penetration and lower MIC for most pathogens. Clinical studies are lacking in non-mycobacterial infections.