Orthogonal experiments of L-9(3(4)) were carried out to select suitable nanofiltration (NF) membrane and optimize operational conditions of NF for advanced treatment of antibiotic wastewater by three commercial membranes (Osmonics, Sepro and Synder Corp). Results showed that the GE membrane was selected through comparing membrane flux and pollutants rejections, and its optimal conditions were at transmembrane pressure (TMP) of 10 bar and pH of 5.0. Under these optimized conditions, the permeate flux reached 64.7 L/h m(2), and the rejection rates of total organic carbon, chemical oxygen demand, UV254, color, and turbidity were 99.3, 91.8, 91.5, 100, and 87.2%, respectively. The rejection rates of multivalent ions were higher than 93%. Humic-like and soluble microbial by-product-like substances in the feed water could be effectively removed. Analysis of variance showed that TMP was the major factor of both permeate flux and monovalent anion removal (p < 0.05), and membrane type was the major factor of removing nearly all pollutants except Na+ and K+ in test conditions. The pH did not have any significant effect on neither permeate flux nor pollutants rejection rates.