Occurrence of non-canonical G-quadruplex (G4) DNA structures in the genome have been recognized as key factors in gene regulation and several other cellular processes. ThemosRandndhA genesinvolved in pathways of oxidation sensing regulation and ATP generation, respectively, makeMycobacterium tuberculosis(Mtb) bacteria responsible for oxidative stress inside host macrophage cells. Circular Dichroism spectra demonstrate stable hybrid G4 DNA conformations ofmosR/ndhADNA sequences. Real-time binding of mitoxantrone to G4 DNA with an affinity constant ~105–107M−1, leads to hypochromism with a red shift of ~18 nm, followed by hyperchromism in the absorption spectra. The corresponding fluorescence is quenched with a red shift ~15 nm followed by an increase in intensity. A change in conformation of the G4 DNA accompanies the formation of multiple stoichiometric complexes with a dual binding mode. The external binding of mitoxantrone with a partial stacking with G-quartets and/or groove binding induces significant thermal stabilization, ~20–29 °C inndhA/mosRG4 DNA. The interaction leads to a two/four-fold downregulation of transcriptomes ofmosR/ndhAgenes apart from the suppression of DNA replication byTaqpolymerase enzyme, establishing the role of mitoxantrone in targeting G4 DNA, as an alternate strategy for effective anti-tuberculosis action in view of deadly multi-drug resistant tuberculosis disease causing bacterial strains t that arise from existing therapeutic treatments.