Eelgrass (Zostera marina L.) is an important organism in coastal marine waters and is highly likely to encounter exposure to multiple stressors, both anthropogenic contaminants and natural stressors. Here, we exposed eelgrass to a range of Cu concentrations and salinities, and also varied exposure route between sediment and water. Measured endpoints were Cu accumulation in root and leaves, relative growth rate, leaf mortality, chlorophyll concentration, and maximum photosynthetic quantum yield. Cu accumulation from the sediment was translocated to all parts of the plant, while Cu taken up from the water showed a tendency to remain in leaves. Effects on relative growth rate and leaf mortality were found only following uptake of Cu from the sediment. We tested effects of different salinities, acting as multiple stressors, together with Cu, but found only weak effects with little interaction with Cu. Experiments with anthropogenic contaminants that marine plants are mainly exposed to through the sediment should be done using sediment exposure, as the common practice of using only water exposure will lead to underestimation of harmful effects. Future studies should take all relevant factors into consideration, as anthropogenic inputs and natural factors are prone to fluctuations due to e.g., climate change.