Fragment-based approaches validate weak-affinity compounds targeting Ldt_Mt2 and Ldt_Mt3 from Mycobacterium tuberculosis. Some fragments inhibit the nitrocefin hydrolysis by by Ldt_Mt2 and Ldt_Mt3. F9 inhibits the growth of Mycobacterium smegmatis and induces morphological alterations in vitro. Combination of biophysical and biological approaches is a plausible strategy to identify fragments that might be starting points for the design of new antituberculosis medicines.