Abstract The defining feature of the mycobacterial outer membrane (OM) is the presence of mycolic acids (MAs), which in part render the bilayer extremely hydrophobic and impermeable to external insults, including many antibiotics. While the biosynthetic pathway of MAs is well studied, the mechanism(s) by which these lipids are transported across the cell envelope is(are) much less known. MmpL3, an essential inner membrane (IM) protein, is implicated in MA transport, but its exact function has not been elucidated. It is believed to be the cellular target of several anti-mycobacterial compounds; however, evidence for direct inhibition of MmpL3 activity is also lacking. Here, we establish that MmpL3 is the MA flippase at the IM of mycobacteria, and is the molecular target of BM212, a 1,5-diarylpyrrole compound. We develop assays that selectively access mycolates on the surface of Mycobacterium smegmatis spheroplasts, allowing us to monitor flipping of MAs across the IM. Using these assays, we establish the mechanism-of-action of BM212 as a potent MmpL3 inhibitor, and employ it as a molecular probe to demonstrate the requirement for functional MmpL3 in the transport of MAs across the IM. Finally, we show that BM212 binds MmpL3 directly and inhibits its activity. Our work provides fundamental insights into OM biogenesis and MA transport in mycobacteria. Furthermore, our assays serve as an important platform for accelerating the validation of small molecules that target MmpL3, and their development as future anti-tuberculosis drugs.