MTX (mitoxantrone) is perhaps the most promising drug used in the treatment of various malignancies. Comprehensive literature on the therapeutics has indicated it to be the least toxic in its class, although its mechanism of action is still not well defined. In the present study, we have evaluated the associated binding interactions of MTX with naked DNA. The mechanism of MTX binding with DNA was elucidated by steady-state fluorescence and a static-type quenching mechanism is suggested for this interaction. Thermodynamic parameters from van 't Hoff plots showed that the interaction of these drugs with DNA is an entropically driven phenomenon. The binding mode was expounded by attenuance measurements and competitive binding of a known intercalator. Sequence specificity of these drug-DNA complexes was analysed by FTIR (Fourier-transform infrared) spectroscopy and molecular modelling studies. CD spectroscopy and the plasmid nicking assay showed that the binding of this drug with DNA results in structural and conformational perturbations. EMSA (electrophoretic mobility-shift assay) results showed that these drug-DNA complexes prevent the binding of octamer TF (transcription factor) to DNA. In summary, the study implicates MTX-induced conformational instability and transcription inhibition on DNA binding.