In this study, multiple roles of biofilm EPS were assessed with respect to the resistance of biofilm and detached biofilm clusters to chlorine disinfection. Strains from an opportunistic pathogen, Pseudomonas aeruginosa (wild type, EPS- and EPS+) with altered extracellular polymeric substances (EPS) secretion capabilities were tested. The impact of biofilm EPS quantity on disinfection was evaluated by monitoring biofilm viability, biofilm structure, removal of dissolved organic matter (DOM), and viability of detached biofilm simultaneously during chlorine disinfection. The obtained results suggested that the presence of EPS increased biofilm and detached biofilm resistance to chlorine in both presence and absence of DOM. The quantity of EPS had an effect on biofilm structure and the structural characteristics were closely related to both overall biofilm viability and the spatial distribution of viable cells within the biofilm. Additionally, the increased amount of EPS influenced selective removal of DOM with polar functional groups. However the DOM removal did not have a significant impact on the viability of biofilm cells during chlorine disinfection. Meanwhile, the viability of detached biofilm clusters, particularly the EPS overproducing strain, was significantly increased in the presence of DOM. The combined results suggested that biofilm EPS played multiple roles toward influencing the resistance of both biofilm and detached biofilm to disinfectant.