Binding of low molecular weight ligands to G-quadruplex nucleic acid structures is known to inhibit telomere extension and prevent abnormal growth of cells leading to cancer. Such specialized DNA conformations have emerged as important targets for drug design and development. In this manuscript we present a detailed calorimetric investigation on the interaction of berberrubine (BBR) and its two synthetic analogues with telomeric G-quadruplex DNA using calorimetry techniques and thermal melting study. A stronger binding of the analogues in comparison with BBR was observed in the experiments. The 13-phenylalkyl derivative of berberrubine (BBR2) (K = 1.38 × 105 M−1) showed higher affinity than the 13-diphenylalkyl derivative of berberrubine (BBR1) (K = 9.70 × 104 M−1). Isothermal titration calorimetry study revealed an exothermic binding that was favoured by both enthalpy and entropy changes in BBR in contrast to the analogues where the binding was mostly enthalpy dominated. A 1:1 binding stoichiometry was revealed in all the systems. The negative standard molar heat capacity values in conjunction with enthalpy–entropy compensation (EEC) confirmed the presence of dominant multiple weak non-covalent interactions in their binding. Differential scanning calorimetry and optical melting study further revealed that BBR and its analogues stabilized quadruplex structure against thermal denaturation. The overall affinity pattern of the alkaloids towards telomeric G-quadruplex structures followed as BBR2 > BBR1 > BBR.