Biofouling is a major drawback for most polyamide reverse osmosis (RO) membranes, which results in water flux decline and higher energy demand. To prevent biofouling, it is important to minimize the interaction between bacterial and membrane surfaces. In this research, we investigated the influence of surface modification with various hydrophilic polymers on biofouling behavior. First, polyamide RO membranes were modified with well-structural controlled hydrophilic polymers, poly(2-hydroxyethyl methacrylate) (pHEMA), poly[poly(ethylene glycol)methacrylate] (pPEG), and poly[(2-methacryloyloxy)ethyl]dimethyl[3-sulfopropyl]ammonium hydroxide (pMEDSAH) via the surface-initiated atomic transfer radical polymerization (SI-ATRP). The biofouling properties were assessed by a static bacterial adhesion and a dynamic biofouling filtration. The membranes modified with a longer main chain of pMEDSAH and a long side chain of pPEG had good resistance against both the bacterial adhesion and dynamic biofouling. Experimental results between the static and dynamic biofouling behavior did not correlate with each other. Molecular dynamics (MD) simulation clarified that pPEG had a longer side chain than pHEMA and pMEDSAH, and that pMEDSAH possessed stronger hydration than pPEG and pHEMA. Experimental results and MD simulation indicated that the main chain length, side chain length, and hydration of the modifying polymer should be taken into account for developing anti-biofouling membranes.