Biophysical cohesive mud, consisting of clay minerals and extracellular polymeric substance (EPS), plays significant role in determining sediments, nutrients and pollutants transport in estuarine and coastal systems. Series of laboratory jar experiments have been conducted aiming at filling the gap of knowledge regarding how biological cohesive EPS affects equilibrium flocculation of EPS-mineral mixtures. Four types of common clay (chlorite, kaolinite, illite and montmorillonite) were chosen due to their abundance in estuarine mud and distinct crystal chemistry and structures. Turbulent shear throughout all the experimental runs were constantly provided at a mean shear parameter of G ≈ 15 s−1 being equivalent to high tidal influenced estuarine turbulent environment. The results reveal that adding EPS increases the equilibrium floc size evidently. The pure mineral flocs show unimodal equilibrium floc size distribution (eFSD) with single peak located at microfloc range (<200 μm) while the EPS-mineral flocs show bimodal eFSD with a secondary peak located in macroflocs range (>200 μm) mostly. Moreover, EPS largely reduces the effective density in EPS-mineral flocs by 1∼2 magnitude. Most importantly, the terminal settling velocity of flocs shows size-dominated in uniform mineral floc cases but density-dominated in EPS-mineral mixture floc cases especially in macroflocs. To model a full floc size or settling velocity distributions in natural environments, furtherly quantification of EPS functions within the large-sized non-fractal mixture floc individually becomes a necessity.