In the present study, a one-pot, green and three-component route for synthesis of Thiocarbohydrazone (TCS) derivatives is described. This reaction was performed in choline chloride/urea (1:2) as Deep Eutectic Solvent (DES). The structures were characterized by FTIR, 1H NMR, 13C NMR spectra and elemental analysis. The derivatives were evaluated for their anti-mycobacterial activity against Mycobacterium bovis BCG, and the results revealed that among the synthesized compounds (1a-1h, 2a-2h, 3a-3h), 3b, 3c and 1b exhibited the highest activity with MIC value of 7.81 and 11.71 μg/mL. Additionally, the target compounds were evaluated for their anti-microbial activity against E.coli and Candida Albicans and the result indicated that in contrast to the low antifungal activity, the produced derivatives with electron-withdrawing substitution at the ortho, meta and para positions of benzyl ring includes 1d, 2b and 3d-3g demonstrated remarkable anti-micobacterial activities.