Mycobacteria are shaped by a thick envelope made of an array of uniquely structured lipids and polysaccharides. However, the spatial organization of these molecules remains unclear. Here, we show that exposure to an esterase from Mycobacterium smegmatis (Msmeg_1529), hydrolyzing the ester linkage of trehalose dimycolate in vitro, triggers rapid and efficient lysis of Mycobacterium tuberculosis, Mycobacterium bovis BCG, and Mycobacterium marinum. Exposure to the esterase immediately releases free mycolic acids, while concomitantly depleting trehalose mycolates. Moreover, lysis could be competitively inhibited by an excess of purified trehalose dimycolate and was abolished by a S124A mutation affecting the catalytic activity of the esterase. These findings are consistent with an indispensable structural role of trehalose mycolates in the architectural design of the exposed surface of the mycobacterial envelope. Importantly, we also demonstrate that the esterase-mediated rapid lysis of M. tuberculosis significantly improves its detection in paucibacillary samples.