The addition of antibacterial material to hollow fiber membranes improves the membrane anti-biofouling characteristics. Antibacterial membranes were fabricated in this study to improve membrane function while also extending membrane lifetime. Neat polyvinylidene difluoride (PVDF) and PVDF hollow fiber membrane with the incorporation of antibacterial agent zinc oxide (ZnO) nanoparticles with various loading (2.5–7.5 wt.%) were fabricated by using dry/wet spinning method. The membrane structure, particle distribution, functional group, hydrophilicity, and pore size of each membrane were all assessed. The result shows that all ZnO/PVDF hollow fiber membranes have the asymmetric structure with even dispersion of ZnO nanoparticles throughout the membranes. The results showed that increased ZnO loadings considerably improved membrane hydrophilicity, and average pore size, in addition to good performance of pure water flux. Antibacterial testing shows that ZnO incorporated in the membrane matrix and membrane surfaces prevents bacteria that cause biofouling from adhering to the membrane. ZnO/PVDF membrane recorded excellent bovine serum albumin (BSA) rejection at 93.4% ± 0.4 with flux recovery rate at 70.9% ± 2.1. These results suggest that antibacterial ZnO/PVDF hollow fiber membranes are promising in relation to reducing biofouling for various water and wastewater treatment.