Systematic thin layer chromatographic (TLC) analysis of apolar lipids in Mycobacterium kansasii revealed the presence of a previously uncharacterized novel component. The product was ubiquitously found in a panel of M. kansasii clinical isolates, as well as other pathogenic and non‐pathogenic mycobacterial species. TLC analysis of [¹⁴C]‐acetate‐ or [¹⁴C]‐glycerol‐labelled M. kansasii cultures tentatively assigned the novel product as an unusual triacylglycerol‐related lipid. Subsequent purification, followed by structural determination using ¹H‐nuclear magnetic resonance (NMR) and electrospray mass spectrometry (ES/MS), led to the identification of this product as a monomeromycolyl‐diacylglycerol (MMDAG). Treatment of M. kansasii with either isoniazid (INH), a well‐known type II fatty acid synthase (FAS‐II) and mycolic acid biosynthesis inhibitor, or tetrahydrolipstatin (THL), a drug approved for treating obesity, correlated with a reduced incorporation of [¹⁴C]‐acetate into both mycolic acids and MMDAG. Addition of INH or THL to the cultures induced major morphological changes and, surprisingly, resulted in an increased number of lipid storage bodies, as determined by electron microscopy. The potent antimycobacterial activity of THL was confirmed against a variety of mycobacterial species, including INH‐susceptible and ‐resistant Mycobacterium tuberculosis strains. Therefore, THL and other β‐lactones may be promising drugs for the development of new antitubercular therapy.