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This paper assessed the collagen glue (Col) from rabbit skin for use as a raw material in combination with different water-based dispersants of antimicrobial agents such as ZnO NPs, TiO2 NPs doped with nitrogen and Ag NPs (TiO2-N-Ag NPs), and chitosan (CS) for the production of biocompatible and antimicrobial nanofibers. The electrospun nanofibers were investigated by scanning electron microscopy (SEM), attenuated total reflectance in conjunction with Fourier-transform infrared spectroscopy (ATR-FT-IR) analyses and antioxidant activity. The biocompatibility of electrospun nanofibers was investigated on cell lines of mouse fibroblast NCTC ((clone L929) using MTT test assays. Antimicrobial activity was performed against Escherichia coli and Staphylococcus aureus bacteria and Candida albicans pathogenic fungus. Electrospun antimicrobial nanofibers based on collagen glue achieved reduction in the number of viable microorganisms against both fungi and bacteria and exhibited multiple inhibitory actions of fungal and bacterial strains. The electrospun nanofibers showed average dimension sizes in the range of 30–160 nm. The results indicated that both Col/TiO2-N-Ag NPs and Col/CS formulations are suitable for cell proliferation and may be useful for producing of nonactive wound dressings.