Biofouling is one of the most important factors that degrade the performance of reverse osmosis (RO) membranes during the desalination process. This study demonstrates that copper ion (Cu(II) or Cu(II) plus hydroxylamine (HA) to produce Cu(I)) in combination with norspermidine (Nspd) can effectively inactivate bacterial biofilms on RO membranes. The addition of Nspd inhibited the inactivation of planktonic P. aeruginosa cells by copper ion. However, with respect to the cells in biofilms (grown in CDC reactors), the use of Nspd enhanced the cell inactivation by copper ion; the addition of Nspd increased the inactivation efficacies of Cu(II) and Cu(II)/HA against biofilm cells from 2.4 to 3.1 and from 1.3 to 3.5 (log inactivation in 1 h), respectively. Nspd disrupted extracellular polymeric substances (as evidenced by the removal of proteins and polysaccharides from biofilms), and it is believed to facilitate the penetration of copper ion into the biofilm matrix. These results showed that the Cu(II)/HA/Nspd treatment also inactivated biofilms in pressurized cross-flow RO filtration units, resulting in partial recovery of permeate flux. However, pretreatment using Nspd (and the subsequent treatment by copper ion) was not as effective as the simultaneous use of Nspd and copper ion in both CDC reactors and cross-flow filtration units.