Zinc oxide nanoparticles (ZnO NP) are well-known for their efficacy as inorganic compounds with antifouling and antibacterial properties in membrane filtration. In this work, bio-zinc oxide nanoparticles (bio-ZnO NP) was synthesized using Aloe vera (Aloe barbadensis miller) powder as a green reducing agent and Zinc nitrate as a precursor through the sol-gel method. Subsequently, the bio-ZnO NP powder was incorporated into a polysulfone (PSf) membrane at varying weight percentages (0.1, 0.3, 0.5, and 1.0 Wt.%). The results indicate that the addition of bio-ZnO NP to PSf enhances the size and complexity of finger-like structures, leading to an increase in tensile strength from 31.9 MPa to 57.6 MPa. However, porosity also increases from 44.1% to 58.2% as the bio-ZnO/NP content rises from 0.1 wt.% to 0.5 wt.%. Notably, the PSf/bio-ZnO NP membrane with 0.5 wt.% concentration demonstrates the highest Pure Water Flux (PWF) at 517.9 LMH with humic acid rejection reaching 99.9%. Furthermore, the incorporation of bio-ZnO NP significantly enhances antibacterial activity, as reflected in the inhibition ring diameter increasing from 24.2 mm to 48.3 mm with higher bio-ZnO/NP concentrations. The PSf/bio-ZnO NP mixed matrix membrane (MMM) with 0.3 wt.% concentration exhibits optimal stability conditions, and superior antifouling properties, making it a promising candidate for membrane applications.