DNA is one of the major molecular targets for a broad range of anticancer drugs. Hence, interaction studies involving cellular DNA and small molecules can be highly beneficial as they often lead to rational and efficient drug design. In this study, the binding interaction of Harmane (a naturally occurring, bioactive indole alkaloid) with two natural polymeric DNAs, that is, Calf thymus (CT) DNA and Herring testis (HT) DNA has been elucidated using biophysical techniques. A ground state, 1:1 complexation, was revealed by steady-state fluorescence spectroscopy. The thermodynamic profile and energetics of the associated reaction were evaluated by temperature-dependent fluorescence spectroscopy. The spontaneity of the binding was confirmed by the negative ΔG° values in both cases. Negative enthalpy change, along with stronger positive entropic contribution, indicated the dominant electrostatic nature of the interaction and finally the entropy-driven exothermic binding process throughout. Salt-dependent studies further demonstrated the significant contribution of electrostatic interactions in ligand binding toward DNA. Infrared data substantiated the structural information of the said interactions, leading to the exploration of the structure-function relationship. © Copyright 2022, Mary Ann Liebert, Inc.