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The in situ formation of silver nanoparticles (AgNPs) aided by chondroitin sulfate and the preparation of a hierarchically structured silver-polymer nanocomposite with antimicrobial activity is shown. Green synthesis of AgNPs is carried out by thermal treatment (80 and 90 °C) or UV irradiation of a chondroitin sulfate solution containing AgNO 3 without using any further reducing agents or stabilizers. Best control of the AgNPs size and polydispersity was achieved by UV irradiation. The ice-segregation-induced self-assembly (ISISA) process, in which the polymer solution containing the AgNPs is frozen unidirectionally, and successively freeze-drying were employed to produce the chondroitin sulfate 3D scaffolds. The scaffolds were further crosslinked with hexamethylene diisocyanate vapors to avoid water solubility of the 3D structures in aqueous environments. The antimicrobial activity of the scaffolds was tested against Escherichia coli . The minimum inhibitory concentration (MIC) found for AgNPs-CS (chondroitin sulfate) scaffolds was ca. 6 ppm.