Zinc oxide (ZnO) nanoparticles are used for the synthesis of various materials. The nanoparticles, when entering into the environment, affect aquatic life. Their antibacterial properties deter the biological treatment process of wastewater treatment plants. The study focuses on the effectiveness of ultrafiltration (UF) membranes for the removal of ZnO nanoparticles. In this study, a commercial membrane was used for the separation of ZnO nanoparticles in the presence of natural organic matter (NOM) and alkalinity. Membrane flux and retention were studied for different concentrations of ZnO (1 mg/L, 10 mg/L, and 100 mg/L). Bare and fouled membranes were studied using a scanning electron microscope (SEM), energy-dispersive X-ray (EDX), and atomic force microscopy (AFM). At higher concentrations (>10 mg/L), ZnO nanoparticles tend to aggregate and increase in size, resulting in 95 to 98% retention. Further, the presence of NOM and alkalinity enhances particle–particle interactions and thereby promotes nanoparticle aggregation, which shows better retention even at lower concentrations (1–10 mg/L).