Gelatin is a biocompatible and biodegradable natural polymer obtained by collagen. Gelatin nanofibers meet all the necessary requirements when used as wound dressing material. However, their lack of antimicrobial properties limits their use. The purpose of this study is to expand the field of use of gelatin by providing it with antimicrobial properties. For this purpose, poly([2-(methacryloyloxy)ethyl] trimethylammonium chloride) (PMETAC), was used. In this study, the polymers were dissolved in formic acid-acetic acid and nanofibers were synthesized by electrospinning. The obtained nanofibers were characterized with SEM, FTIR, and TGA. The antibacterial effect, degradation tests, and cell viability, adhesion and proliferation were investigated. The SEM studies show that the nanofibers are homogeneous and smooth. At the end of 14 days, all nanofibers lost >90% of their mass. The nanofibers containing PMETAC showed good bactericidal activity against Staphylococcus aureus, Escherichia coli, methicillin-resistant Staphylococcus aureus and Acinetobacter baumannii. MTT test demonstrated that low doses of the nanofibers were biocompatible. The cell adhesion study has been shown that many cells attachment and proliferate on the surface of nanofibers. It has been found that the obtained nanofibers can be used safely and effectively as antimicrobial wound dressing material.