The genus Mycobacterium contains two of the most important human pathogens, Mycobacterium tuberculosis and Mycobacterium leprae, the etiological agents of tuberculosis and leprosy, respectively. Other mycobacteria are mostly saprophytic organisms, living in soil and water, but some of them can cause opportunistic infections. The increasing incidence of tuberculosis as well as infections with non-tuberculous mycobacteria (NTM) in immuno-compromised patients has renewed interest in molecular mechanisms of drug resistance in these pathogens. Mycobacteria show a high degree of intrinsic resistance to most common antibiotics. For instance, species from the M. tuberculosis complex (MTC) are intrinsically resistant to macrolides. Nevertheless, some semi-synthetic macrolides as clarithromycin, azithromycin and most recently the ketolides, are active against NTM and widely used for infection treatment. However, shortly after the introduction of these new drugs, resistant strains appeared due to mutations affecting the macrolide target, the ribosome. The mycobacterial cell wall is considered to be a major factor in promoting the natural resistance of mycobacteria to various antibiotics. However, recent data show that specific macrolide resistance determinants (erm genes) are present in some species. This mini-review summarizes the current knowledge on the natural and acquired macrolide resistance in mycobacteria, gives an overview of potential mechanisms implicated in the intrinsic resistance and of macrolide resistance determinants in mycobacteria.