- In tertiary wastewater desalination, effective minimization and cleaning of membrane fouling requires an understanding of the physicochemical interactions between conditioning film–membrane surface, conditioning film–bacteria, bacteria–EPS and within the EPS. In this study, the effect of conditioning film on the attachment of bacteria and EPS (isolated from RO membrane biofouling layers) to the surface was analyzed using quartz crystal microbalance with dissipation (QCM-D) equipped with polyamide-coated sensors. A conditioning film originated from membrane bioreactor (MBR) ultra-filtration permeate (UFP), mainly consisting of effluent organic matter (EfOM). This conditioning film was shown to enhance bacterial attachment but had no effect on the EPS adsorption. Furthermore, the UFP and EPS were characterized and their effects on the swelling behavior of the model polyamide surface were delineated using QCM-D by altering pH values from 6 to 8 and back to 6. In parallel, fouling experiments with UFP and EPS were conducted in a parallel RO plate and frame unit. Reversible changes in the RO permeate flux were observed as pH values were altered (from 6 to 8 and back to 6) with pristine and fouled membrane with EPS. In contrast, irreversible flux decline was observed for the UFP fouled membrane: pH variation, back to 6, promoted further interaction between the UFP organic matter and the membrane, leading to a further reduced membrane permeability. These results are likely related to the swelling phenomenon and its inhibition by the UFP layers observed in the QCM-D. More studies need to be carried out in order to analyze the effects of humic substances, abundant in the UFP fouling layer, that irreversibly interact with the polyamide surface. Reduced membrane swelling capability, which is important for water transport, by different foulants is proposed as additional RO membrane fouling mechanism.