To enhance the antibacterial property and chlorine resistance, organic nanospheres, polypyrrole (PPy), were innovatively incorporated into the polyamide (PA) layer for fabricating thin film nanocomposite (TFN) membranes. Owing to the improved hydrophilicity and compatibility between the PPy nanospheres and the PA layer, the obtained TFN membranes exhibited significant water flux enhancement without sacrificing salt rejection. Under the optimal PPy dosage (0.006 wt%), TFN membranes showed a high water flux of 42.19 L/(m(2) h), which was 118.0% higher than that of the pristine membrane. Remarkably, the chlorine resistance of TFN membranes was strengthened after the incorporation of PPy nanospheres, as evidenced by the less decreased salt rejection, as compared with the TFC and commercial RO membrane. In addition, the positively charged PPy nanospheres immobilized in the PA layer endow the TFN membranes with improved biocidal activity, which was much higher than that of the pristine membrane. This study shows that incorporating organic nanospheres into the PA layer is an effective strategy to develop new ITN membranes with enhanced performance for advanced water purification.