Biofouling on a thin film composite (TFC) membrane is one of the most serious obstacles during the operation of reverse osmosis (RO) processes. In this study, chlorhexidine (CH) was immobilized as a novel non-oxidizing chemical on a RO membrane surface with glutaraldehyde (GA) as a cross-linking agent. The analysis of the membrane surface showed that CH was successfully immobilized with a molecular layer-by-layer technique seen by the increase of Cl and methylene peaks from the XPS and FT-IR, respectively. From the static biofilm growth tests using drip-flow cells, CH immobilized membranes exhibited a high anti-bacterial potential, and the CH bi-layered (CHBL) membrane showed a better anti-biofouling ability than that of the CH mono-layered (CHML) membrane due to the complete coverage of CH on the RO membrane surface. The retardation of biofilm formation and the higher physical cleaning efficiency of the CHML and CHBL membranes were introduced by direct inactivation of the attached bacteria as well as a decreased secretion of biofilm building materials such as exocellular polymeric substances (EPS).