In the treatment of chronic wounds, it is necessary to establish a physiological wound milieu to improve healing. Application of collagen as wound dressing has been described as beneficial as it possesses the ability to reduce elevated levels of proteases, cytokines, and free radicals. Consequently, a wide range of wound dressings based on collagen have been developed. Native collagen is susceptible to alterations because of influences during the production process; to minimize effects on the molecule itself collagen wound dressings are usually aseptically produced. Common sterilization methods (autoclaving, irradiation, and ethylene oxide (EtO) treatment) can induce changes in the protein chemistry and physical properties, potentially affecting the absorption rate, mechanical strength, or performance. In this study, we have evaluated the influence of gamma- and beta-irradiation as well as EtO sterilization on the binding capacity of collagen type I for selected proteases and cytokines associated with nonhealing wounds. Although a pronounced effect on the physical properties of the collagen was found, there was no significant loss in the binding affinity for polymorphonuclear elastase, matrix metalloproteinase-2, and interleukin-1beta, or in the antioxidant capacity.