The fate of engineered nanoparticles in the aquatic-terrestrial transition zone is decisive for their effect in the environment. However, our knowledge on processes within this interface is rather low. Therefore, we used a floodplain stream mesocosm to enhance our understanding of the long-term distribution and biological effects of citrate-coated silver nanoparticles (Ag-NPs) in this ecosystem. Parallel to pulsed dosing of Ag-NPs, we observed fluctuating but successively increasing concentrations of aqueous Ag, 88–97% of which was categorized as particles. The remaining dissolved fraction was mainly complexed with natural organic matter (NOM). The major Ag fraction (50%) was associated with the uppermost sediment layer. The feeding activity of benthic amphipods was largely unaffected, which could be explained by the low Ag concentration and complexation of released Ag⁺ with NOM. According to our hypothesis, only a small nanoparticle fraction (6%) moved to the terrestrial area due to aquatic aging and enrichment of Ag-NPs in sediments and biota. Nanoparticle infiltration in deeper sediment and soil layers was also limited. We expect that a small fraction of nanoparticles remaining in the water for several weeks can be transported over large distances in rivers. The Ag-NPs accumulated in the top layer of sediment and soil may serve as a source of toxic Ag⁺ ions or may be remobilized due to changing physico-chemical conditions. Furthermore, the high enrichment of Ag-NPs on algae (up to 250 000-fold) and leaves (up to 11 000-fold) bears risk for organisms feeding on those resources and for the transfer of Ag within the food web.