A B S T R A C TMicro-organisms were isolated from intake seawater and reverse osmosis (RO) membrane biofilms collected from a full-scale membrane-based desalination process. The results from a culture-dependent approach using 12 media were combined with the microbial community structure on fouled RO membranes as analyzed by a 16S rRNA clone library construction in our previous study. This was followed by selection of 11 target bacteria for further analysis, which were suspected to be responsible for biofilm formation on membrane surfaces. The adhesion of potential biofoulants differing in surface hydrophobicity and charge was examined. Cell wall hydrophobicity was measured as the contact angle of a lawn of bacteria, and by adhesion to hexadecane. The cell surface charge was investigated by measuring electrophoretic mobility. The data obtained from these methodologies were compared. According to the cell surface charge measurements, Pseudomonas aeruginosa, Acinetobacter venetianus, Cellvibrio mixtus subsp. Mixtus, Bacillus sp. Eur1 9.5, and Escherichia coli K12 could mediate initial adhesion to negatively charged RO membranes through electrostatic attraction. Limnobacter sp. KNF002, A. venetianus, and Simiduia agarivorans showed higher affinity to hexadecane than other bacterial strains tested, and Bacillus sp. Eur1 9.5, C. mixtus subsp. Mixtus, and P. aeruginosa were determined to have greater hydrophobic interactions with hydrophobic RO membranes.