The purpose of this study was to prepare various isoniazid derivatives by introducing the isoniazid pharmacophore into several molecules and screening for antimycobacterial activity. Ortho-hydroxy acetophenone reacts with isoniazid to form acid hydrazones. The C-Mannich bases of the above acid hydrazones were prepared by reacting them with formaldehyde and various secondary amines. The synthesized compounds were screened against M. tuberculosis H(37)R(v) using the alamar blue susceptibility test. The synthesized compounds inhibit Mycobacterium tuberculosis strain H(37)R(v) with minimum inhibitory concentrations ranging from 0.56 to 4.61 microM. Compound N'-{1-[2-hydroxy-3-(piperazin-1-ylmethyl)phenyl]ethylidene}isonicotinohydrazide 8 was found to be the most active compound with an MIC of 0.56 microM, and was more potent than isoniazid (MIC of 2.04 microM). After 10 days of treatment, compound 8 decreased the bacterial load in murine lung tissue by 3.7-log10 as compared to controls, which was equipotent to isoniazid. The results demonstrate the potential and importance of developing new isoniazid derivatives against mycobacterial infections.