Biofouling control by quorum sensing (QS) inhibition and the influence of membrane surface characteristics on biofilm formation and QS inhibition were investigated. Pseudomonas putida isolated from the bio-fouled reverse osmosis (RO) membranes in a real plant was used. Acylase was chosen as a model QS inhibitor. Bacteria on the membrane coupons were quantified with the heterotrophic plate count method. Cell distribution was imaged by a confocal laser scanning microscope. Results showed that biofilm formation on the membrane was reduced by acylase as it inhibits the activity of N-acylhomoserine lactone (AHL) which is a signal molecule of QS. It was also shown that membrane surface characteristics were influential factors affecting bacterial adhesion, biofilm formation, and QS inhibition.