This paper aims to elucidate the role of extracellular polymeric substances (EPS) in regulating anion and cation concentrations and toxicity towards microorganisms in anaerobic granular sludges adapted to low (0.22 M of Na+) and high salinity (0.87 M of Na+). The ion exchange properties of EPS were studied with a novel approach, where EPS were entangled with an inert binder (PVDF-HFP) to form a membrane and characterized in an electrodialysis cell. With a mixture of NaCl and KCl salts the EPS membrane was shown to act as a cation exchange membrane (CEM) with a current efficiency of ∼80%, meaning that EPS do not behave as ideal CEM. Surprisingly, the membrane had selectivity for transport of K+ compared to Na+ with a separation factor (SK+/Na+) of 1.3. These properties were compared to a layer prepared from a model compound of EPS (alginate) and a commercial CEM. The alginate layer had a similar current efficiency (∼80%.), but even higher SK+/Na+ of 1.9, while the commercial CEM did not show selectivity towards K+ or Na+, but exhibited the highest current efficiency of 92%. The selectivity of EPS and alginate towards K+ transport has interesting potential applications for ion separation from water streams and should be further investigated. The anion repelling and cation binding properties of EPS in hydrated and dehydrated granules were further confirmed with microscopy (SEM-EDX, epifluorescence) and ion chromatography (ICP-OES, IC) techniques. Results of specific methanogenic activity (SMA) tests conducted with 0.22 and 0.87 M Na+ adapted granular sludges and with various monovalent salts suggested that ions which are preferentially transported by EPS are also more toxic towards methanogenic cells.