In this work we have demonstrated that the ruthenium nitrosyl complex [RuNO(β-Pic)2(NO2)2OH] is suitable for investigation of the inactivation of DNA repair enzymes in vitro. Photoinduced inhibition of DNA glycosylases such as E. coli Endo III, plant NtROS1, mammalian mNEIL1 and hNEIL2 occurs to an extent of ≥90% after irradiation with the ruthenium complex. The photophysical and photochemical processes of [RuNO(β-Pic)2(NO2)2OH] were investigated using stationary and time-resolved spectroscopy, and mass spectrometry. A possible mechanism of the photo-processes was proposed from the combined spectroscopic study and DTF calculations, which reveal that the photolysis is multistage. The primary and secondary photolysis stages are the photo-induced cleavage of the Ru-NO bond with the formation of a free nitric oxide and RuIII complex followed by ligand exchange with solvent. For E. coli Endo III, covalent interaction with the photolysis product was confirmed by UV-vis and mass spectrometric methods.