The present study investigates how dissolved organic matter (DOM) alters copper bioavailability at environmentally relevant concentrations (1–5 μg/L of dissolved copper, 1–4 mg/L of dissolved organic copper). A methodology combining two biological endpoints (short-term and steady-state bioaccumulation of copper by the aquatic moss Fontinalis antipyretica) and a sampling of labile copper with diffusion gradient in thin films (DGT) is proposed for batch experiments conducted with mineral water and various DOM, ethylenediaminetetra-acetic acid (EDTA), humic acid, and natural Seine River (France) extracts (hydrophobic and transphilic fractions). All types of DOM reduce the bioavailability of copper to aquatic mosses, and this reduction was more pronounced for the short-term biological endpoint, which was taken as being representative for environmental exposure. Labile copper sampled with DGT made it possible to estimate short-term bioaccumulation in the case of EDTA and natural Seine River extracts. With humic acid solutions, however, labile copper was lower than bioavailable copper. This result suggests that at realistic metal concentrations and with certain types of natural DOM, bioavailable copper might comprise not only inorganic copper but also some weak organic complexes. Hence, labile copper, in situ sampled with DGT, might not systematically overestimate bioavailable copper, as suggested previously on the basis of in vitro toxicity studies.