The cell wall is a unifying trait in bacteria and provides protection against environmental insults. Therefore, the wall is considered essential for most bacteria. Despite this critical role, many bacteria can transiently shed their cell wall and recent observations suggest a link of such wall-deficient cells to chronic infections. Whether shedding the cell wall also occurs in mycobacteria has not been established unambiguously. Here we provide compelling evidence that a wide range of mycobacterial species, including clinical and non-clinical isolates, form viable cell wall-deficient cells in response to environmental stressors. Using cryo-transmission electron micrography we show that the complex multi-layered wall is largely lost in such cells. Notably, we show that their formation in Mycobacterium marinum and BCG vaccine strains of Mycobacterium bovis is stimulated by exposure to cell wall-targeting antibiotics. Given that these wall-deficient mycobacteria are undetectable using conventional diagnostic methods, such cells have likely been overlooked in clinical settings. Altogether, these results indicate that mycobacteria can readily switch between a walled and wall-deficient lifestyle, which provides a plausible explanation for enabling persistence of infections caused by members of this genus.