<p>Lyocell (Ly) fiber nonwoven fabric is the third generation of regenerated cellulose fiber. It is regarded as an ideal choice in the field of green materials because of its renewable raw materials, environmentally friendly production processes, and biodegradability. However, unmodified fibers lack inherent antibacterial properties, which limits their application in the medical field. Therefore, this study aimed to develop a high-performance antibacterial Ly fiber. The surface activity of Ly fiber was improved by carboxylation with a low eutectic solvent. Then, antibacterial fibers were prepared by grafting a quaternary ammonium salt. Energy-dispersive spectroscopy confirmed the increase in oxygen content and the presence of nitrogen/chlorine. X-ray photoelectron spectroscopy revealed an N1s peak at 398.0&#xa0;eV and a C–N⁺ bond at 396.7&#xa0;eV, directly verifying the grafting of quaternary ammonium salt. The antibacterial test showed that the antibacterial rate of the product against <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> was 99.99%. The cytotoxicity test showed that the product was not cytotoxic. While maintaining the macromorphology of the fiber, the material destroyed the integrity of the membrane through the electrostatic interaction between the positive charge of quaternary ammonium salt and the bacterial cell membrane, realizing high-efficiency antibacterial properties. Thus, quaternary ammonium salt–modified fibers provide a new material basis for the development of medical dressings and functional antibacterial textiles.</p>