Extracellular Polymeric Substances (EPS) in the water environment assemble fine, colloidal particles, such as clays, microorganisms and biomass, in large flocs, which are eventually subject to sedimentation and deposition and determine water/sediment quality and quantity. This study hence aimed to investigate the way that water and colloidal chemistry affects EPS-mediated flocculation of colloidal particles, using a jar-test experiment. Especially, ionic strength, divalent cation and humic substances concentrations were selected as experimental variables in the jar-test experiments, to elucidate their effects on EPS-mediated flocculation. A higher ionic strength increased flocculation capability, reducing electrostatic repulsion between EPS-attached colloidal particles and enhancing particle aggregation. 0.1 M NaCl ionic strength had higher flocculation capability, with 3 times larger floc size and 2.5 times lower suspended solid concentration, than 0.001 M NaCl. Divalent cations, such as Ca2+, built divalent cationic bridges between colloidal particles and EPS (i.e., colloid-Ca2+-EPS or EPS-Ca2+-EPS) and hence made colloidal particles to build into large, settelable flocs. A small Ca2+ concentration enhanced flocculation capability, reducing suspended solid concentration 20 times lower than the initial dosed concentration. However, humic substances, adsorbed on colloidal particles, reduced flocculation, because they blocked EPS adsorption on colloidal particles and increased negative charges and electrostatic repulsion of colloidal particles. Suspended solid concentration in the tests with humic substances remained as high as the initial dosed concentration, indicating stabilization rather than flocculation. Findings about EPS-mediated flocculation in this research will be used for better understanding the fate and transport of colloidal particles in the water environment and for developing the best management practices for water/sediment quality.