Lalistat inhibits growth of Mycobacterium tuberculosis in bacterial culture as well as in infected macrophages, with a minimal inhibitory concentration (MIC) of 25–50 μM. A strong cooperative effect was observed when combined with vancomycin, resulting in a 4-fold MIC drop for lalistat and 16-fold for vancomycin. In infected human macrophages, lalistat reduced bacterial load by 55% compared to the untreated control. Target identification by quantitative proteomics and in situ activity based protein profiling (ABPP) revealed a cluster of 20 hydrolytic proteins including 7 members of the Lip family (LipN, -I, -R, -M, -G, -T and -O). Lipases are essential for M. tuberculosis fatty acid production and energy storage thus representing promising antibiotic targets. We here show that lalistat exhibits an inhibitory effect on M. tuberculosis reproduction and further investigated its mode of action by in situ target identification via ABPP. Several bacterial lipases were identified suggesting inhibition of triacylglycerols (TAGs) hydrolysis as the mode of action. Taken together, the specificity of lalistat for a suite of mycobacterial lipases is intriguing. Given the importance of these enzymes for M. tuberculosis viability during infection they may represent promising drug targets. Future studies need to further dissect and characterize the exact function and mechanism of these enzymes in order to design customized inhibitors suited to interfere with essential metabolic processes specifically in the bacteria. Here, orlistat and lalistat probes represent interesting chemical tools that allow simultaneous and selective detection of lipase activities in living mycobacteria. With regard to the paucity of potent antimycobacterial inhibitors future tuberculosis treatment approaches could largely benefit from adding these compounds into treatment regimens. They could also pave the way for the development of more specific drugs targeting mycobacterial lipases. Their use in combination with known antimycobacterial agents may offer an urgently needed opportunity to improve and shorten TB therapy.