β-resorcylic acid (BR) is a phytochemical which is widely used in the food industry as a flavouring agent and preservative. It has also been found to exhibit antibacterial action against several types of food-borne bacteria. DNA is the main molecular target for many small molecules of therapeutic importance. Hence, the interest is rapidly growing among the researchers to elucidate the interaction between small molecules and DNA. Thus, paving the way to design novel DNA-specific drugs. In this study, an attempt was made to examine the mechanism of binding of BR with calf thymus DNA (ctDNA) with the help of various experiments based on spectroscopy and in silico studies. The spectroscopic studies like UV absorption and fluorescence affirmed the complex formation between BR and ctDNA. The observed binding constant was in the order of 10³ M⁻¹ which is indicative of the groove binding mechanism. These findings were further verified by dye-displacement assay, potassium iodide quenching, urea denaturation assay, the study of the effect of ssDNA, circular dichroism and DNA thermal denaturing studies. Different temperature-based fluorescence and isothermal titration calorimetry (ITC) experiments were employed to evaluate thermodynamic parameters. The analysis of thermodynamic parameters supports the enthalpically driven, exothermic and spontaneous nature of the reaction between BR and ctDNA. The forces involved in the binding process were mainly found to be hydrogen bonding, van der Waals and hydrophobic interactions. The results obtained from the molecular docking and molecular dynamics (MD) simulation were consistent with the in vitro experiments, which support the groove binding mode of BR with ctDNA.