Thioacetazone (TAC) used to be a highly affordable, bacteriostatic anti-TB drug but its use has now been restricted, owing to severe side-effects and the frequent appearance of the TAC resistant M. tuberculosis strains. In order to develop new TAC analogues with fewer side-effects, its target enzymes need to be firmly established. It is now hypothesized that TAC, after being activated by a monooxygenase EthA, binds to the dehydratase complex HadAB that finally leads to a covalent modification of HadA, the main partner involved in dehydration. Another dehydratase enzyme, namely HadC in the HadBC complex, is also thought to be a possible target for TAC, for which definitive evidence is lacking. Herein, using a recently exploited azido naphthalimide template attached to thioacetazone and adopting a photo-affinity based labelling technique, coupled with electrophoresis and in-gel visualization, we have successfully demonstrated the involvement of these enzymes including HadBC along with a possible participation of an alternate mycobacterial monooxygenase MymA. In silico studies also revealed strong interactions between the TAC-probe and the concerned enzymes.