Mycobacterium tuberculosis resistance to commercially available drugs is increasing day by day. To address this issue, various strategies were planned and are being implemented. However, there is a need for new drugs and rapid diagnostic methods. For this endeavour, in this paper, we present the synthesis of acetylene containing 2-(2-hydrazinyl) thiazole derivatives and in vitro evaluation against the H37Rv strain of Mycobacterium tuberculosis. Among the developed 26 acetylene containing 2-(2-hydrazinyl) thiazole derivatives, eight compounds inhibited the growth of Mycobacterium tuberculosis with MIC values ranging from 100 μg ml⁻¹ to 50 μg ml⁻¹. The parent acetylene containing thiosemicarbazones showed promising antimycobacterial activity by inhibiting up to 75% of the Mycobacterium at 50 μg ml⁻¹. In addition, in silico studies were employed to understand the binding mode of all the novel acetylene-containing derivatives against the KasA protein of the Mycobacterium. Interestingly, the KasA protein interactions with the compounds were similar to the interactions of KasA protein with thiolactomycin and rifampicin. Cytotoxicity study results indicate that the compounds tested are non-toxic to human embryonic kidney cells.