ABSTRACT Bacteria have a natural propensity to grow as sessile, matrix-encapsulated, multicellular communities called biofilms. Formation of biofilms proceeds through genetically programmed, distinct developmental stages signaled by intricate networks of communication among the constituent population and their environment. Growing in the complex and heterogeneous microenvironments of biofilms, the resident bacteria acquire unique phenotypes that are generally not associated with their planktonic counterparts. Most notable among these is an extraordinary level of tolerance to a variety of environmental stresses, including antibiotics. Although mycobacteria have long been observed to spontaneously form complex multicellular structures in vitro , it has only recently become apparent that these structures are not only formed through dedicated genetic pathways but are also tolerant to antibiotics. In this article, we review the recent advances in the understanding of mycobacterial biofilms in vitro . We further consider the possible linkage between biofilm-like lifestyles and characteristic persistence of mycobacterial infections against host-defense mechanisms as well as antibiotics.