Nanoparticle (NP) trophic transfer is reported as an important exposure route for organisms in aquatic ecosystems. This study focused on gold nanoparticle (AuNP, PEG-coated, 10 nm diameter) transfer using an experimental benthic food chain which included two trophic levels: natural river biofilm and grazer fish Hypostomus plecostomus. AuNP biofilm accumulation was assessed via water AuNP concentrations and total biofilm mass. An extended range of six AuNP concentrations in water (0, 0.0048, 0.048, 0.48, 4.8, and 48 mg Au L−1) was set. A dose-dependent relation between gold concentrations in water and natural river biofilm was observed after a 48-h exposure. This pointed out the high propensity of natural biofilms to accumulate gold. Additionally, total biofilm mass appeared to influence AuNP accumulation at the highest exposure levels. This first step enables the set-up of the transfer experiment in which grazer fish were exposed for 21 days to natural biofilms, previously contaminated by low AuNP concentrations in water (NP0.1: 0.48 and NP1: 4.8 mg Au L−1). Gold was quantified in eight fish organs, and histology was observed. Gold was transferred from biofilms to fish; bioaccumulation was organ- and exposure level-dependent. Interestingly, the brain showed significant gold accumulation at the highest exposure level (NP1). Histological observations indicated distinct inflammatory responses in fish liver, spleen, and muscle. The overall results suggest the potential hazards of subchronic nanoparticle exposure in aquatic organisms.