Streams are potential receiving compartments for engineered nanoparticles (NP). In streams, NP may remain dispersed or settle to the benthic compartment. Both dispersed and settling NP can accumulate in benthic biofilms called periphyton that are essential to stream ecosystems. Periphytic organisms excrete extracellular polymeric substances (EPS) that interact with any material reaching the biofilms. To understand the interaction of NP with periphyton it is therefore crucial to study the interaction of NP with EPS. We investigated the influence of EPS on the physicochemical properties of selected NP (CeO2, Ag) under controlled conditions at pH 6, 7.6, 8.6 and light or dark exposure. We extracted EPS from five different periphyton communities, characterized the extracts, and exposed CeO2 and carbonate-stabilized Ag NP (0.5 and 5 mg/L, both 25 nm primary particle size) and AgNO3 to EPS (10 mg/L) over two weeks. We measured NP size distribution, shape, primary particle size, surface plasmon resonance, and dissolution. All EPS extracts were composed of biopolymers, building blocks of humic substances, low molecular weight (Mr) acids, and small amphiphilic or neutral compounds in varying concentrations. CeO2 NP were stabilized by EPS independent of pH and light/dark while dissolution increased over time in the dark at pH 6. EPS induced a size increase in Ag NP in the light with decreasing pH and the formation of metallic Ag NP from AgNO3 at the same conditions via EPS-enhanced photoreduction. NP transformation and formation were slower in the extract with the lowest biopolymer and low Mr acid concentrations. Periphytic EPS in combination with naturally varying pH and light/dark conditions influence the properties of the Ag and CeO2 NP tested and thus the exposure conditions within biofilms. Our results indicate that periphytic organisms may be exposed to a constantly changing mixture of engineered and naturally formed Ag NP and Ag+.