Hypothesis: Microbial extracellular polymeric substances (EPS) produced from wastewater are generally heterodispersed, which is expected to influence their flocculation performances and mechanism, particularly in mixed particle systems. The different molecular weight (MW) fractions should contribute to the overall adsorption affinity and flocculation mechanism of EPS in single and dual clay systems. Experiments: EPS harvested from bioreactors were size-fractionated into high, medium and low MW fractions (HMW, MMW, LMW, respectively). The harvested mixed EPS and its fractions were characterised by diverse analytical techniques coupled with optical reflectometry to investigate the role of each EPS fraction in the overall flocculation mechanism of EPS in kaolinite and montmorillonite clay systems. Findings: In single clay systems, both the harvested mixed EPS and the HMW-EPS fraction showed comparable flocculation performances. However, mixed EPS proved to be more efficient than the HMW-EPS fraction for dual clay flocculation. Site blocking effects were observed in mixed EPS: the LMW and MMW EPS first adsorbed to the surface due to higher diffusivities and faster mass transfer to the interface, while the HMW-EPS were slowly transported but were attached to the surface irreversibly and stronger than the LMW/MMW-EPS. We propose from this, a mixed EPS adsorption mechanism: extended anionic polymer tails in solution, thereby enhancing particle flocculation.