The effect of extracellular polymeric substances (EPS), vital organic matters and nutrient elements in the natural environment, on the behavior and toxicology of silver nanoparticles (AgNPs) and ions remains ambiguous. In this study, the role of EPS on the toxicity of AgNPs and dissolved silver ions (from AgNO) to a green algae Chlorella vulgaris was investigated. After the removal of EPS, algae accumulated more silver, about 7.41- and 1.25-fold of those in the algae with EPS for AgNPs and AgNO treatments, respectively. The large amount of accumulated silver was bound to the algal cell surface for AgNPs treatment and was internalized in the algae for AgNO treatment, irrespective of the presence of EPS in algae. After exposure to AgNPs, the ruffles in the surfaces of algal cells were filled by AgNPs, and almost invisible. FTIR showed that for both AgNPs and AgNO, the aldehyde groups on the cell surface were oxidized to carboxyl groups by silver ions, irrespective of the presence of EPS in algal cells, indicating that silver ions were released from the oxidization of AgNPs and reacted with algal cells. The content of chlorophyll showed that AgNPs depressed algal growth more remarkably than did AgNO, independent of the presence of EPS in algae, suggesting that AgNPs had greater toxic effects on algae than did silver ions. The findings suggest that the barrier effect of EPS gave nanoparticles an extraordinary edge over ions, but EPS had no discerning effect on the interaction of algal cells with the silver ions released from AgNPs and AgNO, and also on the effect of AgNPs and AgNO on algal growth.