The effects of polysaccharides composition on the interactions between EPS and RO membrane, including fouling, were studied in the presence of Ca2+ EPS originated from Pseudomonas aeruginosa PAO1 biofilms of the wild type and its Apsl isogenic mutant was used for RO membranes fouling and QCM-D adsorption experiments. For the wild type strain EPS, bridging of alginates by Ca2+ led to an increase in both adsorption and rigidity of the adsorbed layer. However, no change was detected for the Delta sIEPS adsorption and the obtained layer showed reduced rigidity, likely due to the interference of Ca2+ with interactions between the polysaccharides composing the EPS. In agreement with the QCM-D results, once Ca2+ was added, an increased in RO fouling was observed with the wild type EPS, while reduced fouling was found for the Delta psl mutant EPS. The presence of an ATR-FTIR peak characterized glycosidic linkage only for the fouling layer formed by the EPS of the 451 mutant in the presence of Ca2+, suggested that Ca2+ alters the scaffold matrix of this EPS. The results of this study showed that polysaccharide composition in the EPS matrix significantly influences the interaction with Ca2+ as well as membrane fouling.