Feeding cadmium (II) and selenium (IV) simultaneously to anaerobic granular sludge with the aim of synthesizing cadmium selenide (CdSe) nanoparticles induces compositional changes in the extracellular polymeric substances (EPS) matrix of this sludge. A methanogenic anaerobic granular sludge was repeatedly exposed to Cd(II) (10-50 mg L-1) and selenite (79 mg L-1) for 300 days at pH 7.3 and 30 degrees C in a fed-batch feeding regime for enrichment of Se-reducing bacteria and synthesis of CdSe nanoparticles. EPS fingerprints of the granular sludge, obtained by size exclusion chromatography coupled to a fluorescence detector, showed a significant increase in the intensity of protein-like substances with > 100 kDa apparent molecular weight (aMW) upon repeated exposure to Cd(II) and Se(VI). This was accompanied by a prominent decrease in protein-like substances of aMW < 10 kDa. The fingerprint of the humic-like substances showed emergence of a new peak with aMW of 13 to 300 kDa in the EPS extracted from the Cd/Se fed granular sludge. Experiments on metal(loid)-EPS interactions showed that the CdSe nanoparticles interact mainly with loosely bound EPS (LB-EPS). This study showed that the formation of Se(0) and CdSe nanoparticles occurs in the LB-EPS fraction of the granular sludge and repeated exposure to Cd and Se induces compositional changes in the EPS matrix.