In this study, benzyl triethylammonium chloride (BTEAC)-functionalized poly(vinyl alcohol) (PVA) nanofibers were fabricated via electrospinning and deposited on a commercial polycarbonate (PC) membrane (f-PVA/PC membrane) in order to enhance the anti-biofouling activity of the membrane. Permeation flux, biofilm formation, contact, and regeneration tests were performed to evaluate the anti-biofouling potential of the f-PVA/PC membrane against bacteria, including Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. The permeation flux test showed that the normalized flux of the f-PVA/PC membrane was retained at 1.0 after filtration of 500 mL of the bacterial solution (K. pneumoniae), whereas the fluxes of the PC and PVA/PC membranes decreased to 0.57 and 0.84, respectively. In the biofilm formation test, the number of biofilm cells on the f-PVA nanofibers (P. aeruginosa = 4.21-4.98 log colony-forming unit (CFU), S. aureus = 3.74-4.39 log CFU) was less than those on the PVA nanofibers (P. aeruginosa = 5.68-6.89 log CFU, S. aureus = 4.72-5.82 log CFU). The contact test demonstrated that mortality rates (contact time = 60 min) on the f-PVA/PC membrane (K. pneumoniae = 94.08%, S. aureus = 99.99%, E. coli = 92.30%) were greater than those on the PC membrane (K. pneumoniae = 73.75%, S. aureus = 62.41%, E. coli = 76.80%). Fluorescence microscopy images illustrated that the population of red (dead) bacterial cells on the f-PVA/PC membranes was greater than that on the PVA/PC membrane. The regeneration test indicated that the f-PVA/PC membrane retained its anti-biofouling activity during regeneration and reuse over six cycles with mortality rates of 93.82-96.29% (K. pneumoniae), 74.27-85.15% (S. aureus), 91.68-95.19% (E. coli), and 94.94-96.90% (mixed-strain bacteria). The results demonstrated that the f-PVA nanofibers could enhance the anti-biofouling potential of the membrane through both anti-adhesive and anti-bacterial surface modifications.