A series of novel isonicotinoyl hydrazones based on pyridoxine (vitamin B₆) were synthesized. The synthesized compounds were evaluated for their antimycobacterial activity on M. tuberculosis H₃₇Rv strain. The most potent compound 13 showed good activity on H37Rv strain and on clinical isolates of M. tuberculosis with multidrug-resistant tuberculosis (TB) profile included first- and second-line drugs. Cytotoxicity studies of compound 13 on human embryonic kidney cells, human liver, human mesenchymal stem cells, and human embryonic lung cells in vitro demonstrated it is 2–3 times less toxicity then isoniazid and 1.5–2 less toxicity than ethambutol and moxifloxacin. Compound 13 showed weak complexation with Fe³⁺ ions, low acute toxicity (LD₅₀ > 2000 mg/kg per os on mice) and the identical to isoniazid and significantly better than ethambutol and moxifloxacin efficacious in the mouse model of drug-sensitive (H37Rv) TB. These facts make him a promising candidate for future developments of antitubercular drugs. [Figure not available: see fulltext.].