DNA interactions with multivalent ligand(s) have increasingly become the subject of substantial research. For several small molecules with therapeutic potential, nucleic acids serve as their primary molecular target. Such interaction has been shown to affect transcription or replication, ultimately leading to apoptotic cell death. As a result, researchers are becoming increasingly interested in understanding how small molecules interact with DNA making it possible to develop new, DNA-specific drugs. The bioactive indole alkaloid, Yohimbe (Yohimbine; Yh) has been broadly studied in pharmacological properties while its binding mode to DNA has not been explicated so far. This study adopted molecular modelling and multi-spectroscopic methods to investigate the interaction between Yohimbine and herring testes (HT DNA) in physiological conditions. Minor hypochromic and bathochromic shifts of fluorescence intensity were observed, suggesting the binding of Yh to HT DNA. The Scatchard plot analyses using the McGhee-von Hipple method revealed non-cooperative binding and affinities in the range of 10⁵ M⁻¹. The thermodynamic parameters suggested exothermic binding, which was favoured by negative enthalpy and positive entropy changes from temperature-dependent fluorescence experiments. Salt-dependent fluorescence suggested that the interaction between the ligand and DNA was governed by non-polyelectrolytic forces. The results of iodide quenching, urea denaturation assay, dye displacement, and in silico molecular docking, suggested groove binding of Yh to HT DNA. Thus, the groove binding mechanism of interaction was validated by both biophysical and computational techniques. The structural elucidation and energetic profiling of Yh's interaction with naturally occurring polymeric DNA can be useful to the development of DNA-targeted therapeutics.