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Microbial infections due to bacteria, viruses, and molds are a serious threat to both human life and the health of other organisms. To develop inexpensive, easy-to-prepare, efficient, and portable nano-antibacterial materials, as well as to explore the antibacterial prospects of cationic antibacterial agents, in this work, six different membrane materials were prepared by the electrostatic spinning method and characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier transform infrared (FT-IR). The materials were tested for antimicrobial properties using a modified AATCC100-200 test method. Under the most suitable spinning conditions, the doping amount of the cationic antimicrobial agent, CTAB, had the greatest influence on the antimicrobial performance. The antimicrobial performance of PCL/PEO/CS/CTAB0.4 was the highest among the prepared materials, with 83.7% effectiveness against S. aureus and 99.9% against E. coli. The antimicrobial performance was found to be stable. In our study, we determined the most suitable spinning ratio to prepare an inexpensive and efficient cationic antimicrobial agent. Biodegradable, high-antimicrobial-activity antimicrobial materials can be applied as films, and this new nanofiber material has shown great potential in wound dressings and as a mask material due to its remarkable antimicrobial efficiency.