The use of bioactive proteins such as keratin has been successfully explored to improve the biological interface of scaffolds with cells during the tissue regeneration. In this work, it is optimized the fabrication of nanofibers combining wool keratin extracted by sulfitolysis, with polycaprolactone (PCL) in order to design bicomponent fibrous matrices able to exert a selfâadapting pattern of signalsâmorphological, chemical, or physicalâconfined at the single fiber level, to influence cell and bacteria interactions. It is demonstrated that the blending of highly polydisperse keratin with PCL (50:50) improves the stability of the electrospinning process, promoting the formation of nanofibersâ144.1â±â43.9 nmâwithout the formation of defects (i.e., beads, ribbons) typically recognized in the fabrication of keratin ones. Moreover, keratin drastically increases the fiber hydrophilicityâcompared with PCL fiber aloneâthus improving the hMSC adhesion and in vitro proliferation until 14âdays. Moreover, the growth of bacterial strains (i.e., Escherichia coli and Staphylococcus aureus) seems to be not specifically inhibited by the contribution of keratin, so that the integration of further selected compounds (i.e., metal ions) is suggested to more efficiently fight against bacteria resistance, to make them suitable for the regeneration of different interfaces and soft tissues (i.e., skin and cornea). © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1803â1813, 2019.