Here, a medicinal chemistry study of an N-alkylphenyl-3,5-dinitrobenzamide (DNB) scaffold as a potent anti-TB agent is presented. A series of chemical modifications were performed and forty-three new molecules were synthesized to study the structure–activity relationship (SAR) by evaluating against a sensitive strain (H37Rv) of Mycobacterium tuberculosis (MTB). Potent DNB analogs 4b, 7a, 7c, 7d, 7j, 7r and 9a were further tested against resistant strains of MTB. Their intracellular as well as bactericidal potential was also evaluated. Cytotoxicity and in vivo pharmacokinetic studies suggested that DNB analogs have an acceptable safety index, in vivo stability and bio-availability. From the present work, two compounds 7a and 7d have shown nanomolar to sub micro-molar MIC in extracellular and intracellular assays.