Mycobacterial species, including Mycobacterium tuberculosis, rely on lipids to survive and chronically persist within their hosts. Upon infection, opportunistic and strict pathogenic mycobacteria exploit metabolic pathways to import and process host-derived free fatty acids, subsequently stored as triacylglycerols in the form of intrabacterial lipid inclusions (ILI). Under nutrient-limiting conditions, ILI constitute a critical source of energy that fuels the carbon requirements and maintain redox homeostasis, promoting bacterial survival for extensive periods of time. In addition to their basic metabolic functions, these organelles display multiple other biological properties, emphasizing their central role in the mycobacterial life cycle. However, despite their importance, the dynamics of ILI metabolism and their contribution to mycobacterial adaptation/survival in the context of infection has not been thoroughly documented. Herein, we provide an overview of the historical ILI discoveries, their characterization and current knowledge regarding the microenvironmental stimuli conveying ILI formation, storage and degradation. We also review new biological systems to monitor the dynamics of ILI metabolism in extra- and intracellular mycobacteria and describe major molecular actors in triacylglycerol biosynthesis, maintenance and breakdown. Finally, emerging concepts regarding the role of ILI in mycobacterial survival, persistence, reactivation, antibiotic susceptibility and inter-individual transmission are also discussed.