In recent years, microbial fermentation has become a sustainable alternative to traditional petrochemical processes for producing biomass nylon 56 (i.e., PA56). This study is centered on creating a highly efficient antibacterial nanofiber membrane using bio-nylon 56 as the main material. The membrane was fabricated via a multi-step process involving sodium alginate, chitosan, and poly(hexamethylene biguanide) (PHMB). The PA56 nanofiber was chemically modified by sequential coupling with alginate (AG) and chitosan (CS), introducing a significant number of functional groups (-COOH and -NH2). This process resulted in the formation of PA56-AG and PA56-AG-CS nanofibers. Further modification with PHMB led to obtaining the PA56-AG-PHMB and PA56-AG-CS-PHMB antibacterial nanofiber membranes. The optimal preparation conditions for these membranes were determined, including the pH and concentration of AG, the molecular weight, pH, and concentration of CS, and the pH and concentration of PHMB. The PA56-based membranes demonstrated nearly 100 % antibacterial efficiency within a short time. However, the PA56-AG-PHMB membrane exhibited faster antibacterial rates and higher efficiency in repeated use compared to the PA56-AG-CS-PHMB membrane. The two-step coupling reaction in the preparation of PA56-AG-CS-PHMB may have reduced its surface accessibility to E. coli cells, resulting in slower bacterial attachment. Furthermore, the PA56-related membranes showed excellent biocompatibility, with a 100 % cell survival rate. Despite some limitations in reusability, biomass nylon PA56 stands out as an environmentally friendly material derived from renewable resources through microbial fermentation. It offers significant sustainability advantages over traditional petroleum-based nylons, as evidenced by the favorable cytotoxicity test results.