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In this study, the employment of the X-ray irradiation as bioremediation method to treat parchment has been deeply investigated. In particular, the effect of the irradiation doses on the structural stability of collagen, the main constituent of parchment, has been evaluated on a series of modern parchment samples by means of different opto-thermal and spectroscopic techniques in order to obtain the dose-dependent effect of irradiation on collagen. Moreover, the long-term behavior of the irradiated parchment has been considered by analyzing the same series of samples after being hygrothermally artificially aged. Characterizations by light transmission analysis, Fourier transform infrared spectroscopy and the ultraviolet–visible–near infrared (UV–Vis–NIR) reflectance one have been performed for the identification of the radio-induced effect on the parchment structure even in a dose range much wider than the need for sterilization. The obtained results have proved the safeness of the method in the short and long term confirming the applicability of this emerging procedure.