The present study demonstrates a detailed spectroscopic investigation on the differential extent and modes of interaction of two alkaloid drugs, Sanguinarine (SG) and Berberine (BR), with biologically significant protonated and B-forms of herring sperm DNA in aqueous buffer medium of pH 3.5 and 7.4 respectively. The steady-state spectroscopic results substantiate the binding interaction of either drug molecule with protonated as well as B-form of DNA. The drug:DNA binding strength obtained employing the Benesi-Hildebrand equation reveals a significantly higher binding affinity of BR with B-DNA at pH 7.4 compared to that with protonated DNA at pH 3.5. However, the binding constant obtained for interaction of iminium form of SG with protonated DNA is found to be an order of magnitude higher compared to that in case of interaction with B-DNA. Moreover, the steady-state emission results validate the role of preferential electrostatic interaction between the cationic forms of the drugs (cationic BR and the iminium form of SG) and the negatively charged phosphate backbone of B-DNA under physiological conditions at pH 7.4 which has been further corroborated by monitoring the effect of a strong electrolyte on drug:DNA interaction under the various experimental conditions. The circular dichroic (CD) spectroscopic studies reveal the probable intercalative binding of the drug molecules within DNA at either pH. The time-resolved fluorescence decay measurements along with the time-resolved fluorescence anisotropy studies further substantiate the binding interaction of the drugs with protonated as well as B-form of herring sperm DNA. © 2020 Elsevier B.V.