The Actinobacteria produce an array of valuable metabolites including biosurfactants which are gaining increased attention in the biotechnology industries as they are multifunctional, biorenewable and generally superior to chemically synthesized compounds. Biosurfactants are surface-active, amphipathic molecules present at the microbial cell-surface or released extracellularly and in a variety of chemical forms. The mycolic acid-containing actinobacteria (MACA), classified in the order Corynebacteriales, represent a potentially rich source of biosurfactants for novel applications and undiscovered biosurfactant compounds. Members of the mycolate genus Rhodococcus produce various well-characterised glycolipids. However, other mycolate genera including Corynebacterium, Dietzia, Gordonia and Tsukamurella although less extensively investigated also possess biosurfactant-producing strains. This chapter captures current knowledge on biosurfactant production amongst the MACA, including their chemical structures and producer organisms. It also provides an overview of approaches to the recovery of biosurfactant producing MACA from the environment and assays available to screen for biosurfactant production. Methodologies applied in the extraction, purification, and structural elucidation of the different types of biosurfactants are also summarised. Potential future applications of MACA-derived biosurfactants are highlighted with particular focus on biomedical and environmental possibilities. Further investigation of biosurfactant production by MACA will enable the discovery of both novel producing strains and compounds with the prospect of biotechnological exploitation.