An array of pyridine appended 2-hydrazinylthiazole derivatives has been synthesized to discover novel chemotherapeutic agents for Mycobacterium tuberculosis (Mtb). The drug-likeness of pyridine appended 2-hydrazinylthiazole derivatives was validated using the Lipinski and Veber rules. The designed thiazole molecules have been synthesized through Hantzsch thiazole methodologies. The in vitro antimycobacterial studies have been conducted using Luciferase reporter phage (LRP) assay. Out of thirty pyridine appended 2-hydrazinylthiazole derivatives, the compounds 2b, 3b, 5b, and 8b have exhibited good antimycobacterial activity against Mtb, an H37Rv strain with the minimum inhibitory concentration in the range of 6.40–7.14 μM. In addition, in vitro cytotoxicity of active molecules has been observed against Human Embryonic Kidney Cell lines (HEK293t) using MTT assay. The compounds 3b and 8b are nontoxic and their cell viability is 87% and 96.71% respectively. The in silico analyses of the pyridine appended 2-hydrazinylthiazole derivatives have been studied to find the mode of binding of the active compounds with KasA protein of Mtb. The active compounds showed a strong binding score (−5.27 to −6.23 kcal mol⁻¹).