The present study is aimed at probing the molecular interaction between Carmustine (anti-cancer drug) and Melatonin (hormone) within the realm of Density functional theory at B3LYP/6–31 G (d,p) level, and using vibrational spectroscopic techniques, namely, FTIR (400–4000 cm–1), Raman (145–3560 cm–1) and surface-enhanced Raman spectroscopy (SERS) (145–3560 cm–1) techniques as well. The vibrational spectra are assigned for both monomers and the interacting complex incorporating the VEDA program and found to be consistent with the experimental findings. NBO analysis has been performed to explore the charge transfer path between Carmustine and Melatonin that provides a reliable insight into the origin of N–H∙∙∙O intermolecular hydrogen bond, which is further supported by the MEP analysis. The complex's thermodynamic and quantum chemical parameters are computed and compared to the respective values of the individual states. The biomolecular complex is found to have low HOMO-LUMO gap and high hyper-polarizability values indicating the complex to be high chemical reactivity and nonlinear optical activity. Moreover, molecular docking analysis is performed to investigate the pharmaceutical activeness of the complex against the selected MIA protein.