Mycolic acids are one of the basic elements of the cell wall structure of bacteria belonging to the suborder Corynebacterineae, constituting from 20% to 40% of dry weight. Additionally, they show high structural diversity within each family and species. Nowadays, profiles of mycolic acids are widely described for the genus Mycobacterium, the causative agent of tuberculosis. However, the suborder Corynebacterineae also includes many representatives of opportunistic human pathogens, e.g. Dietzia, Gordonia, Nocardia and Rhodococcus. Currently, an increased infection risk caused by this group of microorganisms especially in immunocompromised patients has been observed. Better knowledge of mycolic acid profiles for Corynebacterineae may allow identification of mycolic acids as diagnostic markers in the detection of opportunistic bacterial infections. Modern techniques of chemical analysis, including mass spectrometry, may enable the development of new chemotaxonomic methods for the detection and differentiation of bacteria within the suborder Corynebacterineae.