The interaction of a series of potent leishmanicidal aromatic diamidines resembling pentamidine, was studied with Leishmania infantum DNA and polynucleotides. The diamidines viz., CGP040215A, CGP033829A and CGP039937A, interacted with leishmania DNA as well as with the polynucleotides poly(dA)-poly(dT), poly(dA-dT) and poly(dG-dC). The thermodynamic analysis to determine the association constants and the binding enthalpy pointed toward binding of the diamidines at AT regions of the DNA. The results also indicate that the diamidines bind at the outside of the DNA double helix, probably to the minor groove regions, with hydrogen bonds connecting the amide nitrogen of the diamidine to carbonyl oxygen atoms of thymidine or adenosine bases. However, CGP040215A and CGP033829A, the bisaryl diamidines, showed higher affinity than CGP039937A, the monoaryl diamidine. The spectrophotometric analysis of the interaction of these diamidines to test their effects on the melting temperature of leishmanial DNA suggests non-intercalating binding. The diamidines also showed potent inhibition of DNA polymerase activity of L. infantum extracts in vitro.