Novel 2-[3-(4-bromo-2-fluorobenzyl)-4-oxo-3,4-dihydro-1-phthalazinyl]acetic acid hydrazones were synthesized from phthalic anhydride by a six step synthesis and evaluated for in vitro, in vivo activities against eight mycobacterial species and Mycobacterium tuberculosis (MTB) isocitrate lyase (ICL) enzyme inhibition studies. Among twenty six compounds N'1-[(4-nitrophenyl)methylene]-2-[3-(4-bromo-2-fluorobenzyl)-4-oxo-1,2,3,4-tetrahydro-1-phthalazinyl]ethanohydrazide (7j) was found to be the most active compound in-vitro with MIC's of 0.18 and <0.09 microM against log-phase cultures of MTB and multi-drug resistant MTB respectively. Compound 7j inhibited all the eight mycobacterial species with MIC ranging from <0.09-12.25 microM and was not toxic to Vero cell lines till 122.5 microM. Seven compounds were tested against starved culture of MTB and they inhibited with MIC's ranging from 2.88-8.91 microM. Some compounds showed 45-61% inhibition against MTB ICL enzyme at 10 microM. In the in vivo animal model 7j decreased the bacterial load in lung and spleen tissues with 1.87 and 3.03-log10 protections respectively at 25 mg/kg body weight dose.