Cellulose acetate (CA) nanofibers have been popularly applied in various biomedical and textile products. In this work, a textile azo-dye Reactive Green 19 (RG19) was selected to be chemically coupled to the CA nanofiber membrane to form dyed CA nanofiber membrane (namely CA-RG19) and then poly(hexamethylene biguanide) (PHMB) as an antibacterial reagent was physically attached to the dyed CA nanofiber membrane, forming CA-RG19-PHMB nanofiber membrane. The nanofiber membranes were evaluated for their physical and mechanical properties, including functional group analysis, morphological characterization, and thermal stability assessment. To investigate the antibacterial properties of the nanofiber membrane, various concentrations of RG19 dye and PHMB were tested to evaluate the antibacterial efficiency (AE) against Escherichia coli of the membranes. It was found that the CA-RG19-PHMB nanofiber membrane exhibited an AE value of approximately 100 %, with the immobilization concentrations of RG19 dye and PHMB being 373.46 mg/g and 0.333 mg/g, respectively. The CA-RG19-PHMB nanofiber membrane showed 100 % antibacterial efficacy after 10 min against E. coli cells. Furthermore, the storage stability of the CA-RG19-PHMB nanofiber membrane remained at approximately 100 % of its initial antibacterial efficacy after 60 days, and it exhibited excellent antibacterial efficacy after five cycles.