Background and Objectives: The objective of this study is to synthesize and evaluate 1-(4-chlorophenyl)-4-(4-hydroxy-3-methoxy-5-nitrobenzylidene) pyrazolidine-3,5-dione (CD59) analogues to establish structure-activity relationship and mechanism of action., Methods: Thirty analogues of reported antitubercular CD59 were prepared by two-step synthetic protocols and characterized. The compounds were evaluated for in vitro activities against Mycobacterium tuberculosis (MTB), cytotoxicity against RAW 264.7 cells. The molecules were also evaluated for three mycobacterial enzymes to study the mechanism of action., Results: Among the compounds, 4-(2-bromobenzylidene)-1-(4-chlorophenyl)pyrazolidine-3,5-dione (4k) was found to be the most active compound in vitro with MICs of 4.13μM against log-phase culture of MTB and also non-toxic up to 50μM., Conclusions: Amongst all, the compounds 4g, 3i and 3n were most active against the enzymes MTB Pantothenate synthetase, lysine amino transferase and Alanine dehydrogenase, respectively. Further screening of these molecules was required in the in vitro dormant MTB models., (Copyright © 2014 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.)