Development of fouling layer on reverse osmosis (RO) membrane significantly harm the performance of membrane. Among various foulants, aromatic protein-like substances were remained on the membrane after the chemical cleaning and speculated to have not only electrostatic or hydrophobic interactions but also have strong quadrupole interactions like cation-π and π-π interactions with membrane. Even though previous study analyzed irreversible foulants in protein level, to better understand fouling mechanism, peptides level analysis was conducted. By using E.coli K-12 as model bacteria, biofilm was developed through the continuous RO operation and peptides in extracellular polymeric substances were analyzed through LC-ESI/MS/MS. Among peptides analyzed, ompC protein peptide (INLLDDNQFTR) enhanced irreversibility most significantly and atomic force microscopy (AFM) analysis showed that the adhesion force of INLLDDNQFTR against RO membrane was the strongest. By analyzing adhesion force of peptides and membrane at pH 4 and 7, attractive force such as cation-π and π-π interactions were major. To control the irreversible foulants, ionic liquid (IL) which has strong solvation properties by cation-π and π-π interactions with solutes, was utilized. In total 9 types of IL were compared and cleaning efficiency of [BPy][Triflate] was the highest, and the interactions between foulants-foulants and foulants-membrane analyzed by AFM in the presence of [BPy][Triflate] became the weakest. Optical coherence tomography showed that significant swelling was induced in the fouling layer when [BPy][Triflate] was introduced. Specifically, cation parts of IL had cation-π and π-π interactions and anion parts IL had hydrogen bonding with organic foulants, and thus fouling layer became swelled. In the feasibility test of ionic liquid-based cleaning agents, the addition of IL to the alkaline cleaning solution mitigated the irreversible fouling phenomenon without deteriorating membrane performance for long-term operation. Economic feasibility test showed that addition of ionic liquids to the alkaline solution was economically feasible by extending chemical cleaning period.