The environmental fate of silver nanoparticles (nAg) in freshwater is strongly influenced by interactions with biogenic organic matter, particularly extracellular polymeric substances (EPS) released by phytoplankton. In the present study, the effect of EPS released by green alga Chlamydomonas reinhardtii on aggregation kinetics of nAg related to mono- and di-valent ions was examined. The research approach involved state-of-the-art analytical techniques (dynamic light scattering, asymmetric field flow-field fractionation coupled with fluorescence detector, UV-Vis absorbance) and Derjaguin–Landau–Verwey–Overbeek (DVLO) theory. The results demonstrated that the adsorption of EPS altered nAg surface properties, enhancing their stability and reducing aggregation, thereby increasing persistence and distribution in the water column. In presence of EPS, the critical coagulation concentration (CCC) of nAg increased markedly for both mono- and divalent ions compared to EPS-free conditions: for Na⁺ the CCC increased from 100 to 450 mM, while for Ca²⁺ from 0.7 to 4.6 mM. Extended DVLO theory simulations supported these observations, showing that the formation of an EPS-derived ecocorona introduces steric repulsion that increases the energy barrier to aggregation. The colloidal stability of nAg in presence and absence of EPS was further evaluated in synthetic freshwater, confirming that EPS-induced transformations consistently reduced aggregation of nAg under more realistic conditions. The findings of the present study demonstrate that EPS-mediated ecocorona formation plays a critical role in governing the stability, transport, and environmental fate of nAg, with important implications for their distribution, persistence, and ecological footprint in aquatic environments.