The potential impact of graphene oxide (GO) on the toxicity of metals in complex mixtures on freshwater species at ecologically relevant exposure levels was studied. The behavioural responses of brown trout (Salmo trutta) larvae and juveniles to single and combined exposure of GO and four common metals (zinc, copper, nickel, and chromium) in a complex mixture (MIX) were investigated. Fish larvae were exposed in groups for 1 and 4 days, and juveniles were treated individually in open-field tanks for 2 hours with single and combined exposures of GO (1, 10, and 20 mg L⁻¹) and MIX (Zn²⁺ – 100, Cu²⁺ – 10, Ni²⁺ – 34, and Cr^3+/6+ – 10 μg L⁻¹) solution. MIX was prepared in compliance with the maximum-allowable-concentrations (MAC) for certain metals in European rivers and lakes. Fish larvae in nests were reduced up to 12.4 ± 2.0% after a single GO exposure, whereas MIX exposure reduced the response up to 19.7 ± 5.3% at the highest concentrations examined after 1 and 4 days of exposure. Exposure to MIX with added GO showed that fish larvae were less affected when exposed to a single MIX. Fish juveniles exposed to combined GO and MIX showed opposite responsive tendencies, where the interaction between GO and MIX intensified the trace element effects by significantly increasing (from 1.8 to 2.8 fold) swimming activity in juveniles compared to those exposed to MIX. These findings show that GO modulates the toxicity of metal pollutants in complex mixtures on a biological scale.