The present work focuses the investigation on fluorescence quenching of nile blue (NB) in presence of various substituted uracil molecules. UV–Visible absorption studies signify the possibility of ground state complex formation between NB and uracil molecules. The increase in concentration of quencher molecules greatly influences the emission spectra of NB. The bimolecular quenching rate constant (kq) were calculated and found to depend on the position and electronic properties of substituent in quencher molecules. Fluorescence quenching experiments were performed at different temperature to calculate the thermodynamic parameters. The fluorescence lifetime measurements show that the quenching process proceeds through static quenching. The mechanism of fluorescence quenching includes the possibility of proton transfer. The bond dissociation enthalpy (BDE) reveals the release of H[rad] from the quencher molecules. The quencher molecules possess antioxidant activity and identified using deoxyribose degradation assay. The position of substituent and its electronic property are key features to address the antioxidant activity of uracil molecules. © 2020 Elsevier B.V., All rights reserved.