Abstract As the use of products incorporating plastics and metal-based nanoparticles (NPs) continues to rise, their co-existence in aquatic environments is becoming increasingly common. This raises the potential for interactions between the two, which may in turn influence the behavior, bioavailability, and toxicity of metal-based NPs. In this study, we explored how polystyrene microplastics (PS-MPs) and PS nanoplastics (PS-NPs) affect the toxicity and action mechanisms of copper oxide NPs (CuO-NPs) in Daphnia magna. Our results demonstrated that both PS-NPs and PS-MPs significantly enhanced the toxicity of CuO-NPs to D. magna, reducing the median lethal concentration values from 4.007 mg L−1 (in the absence of PS-MPs/NPs) to 3.156 mg L−1 and 1.727 mg L−1, respectively. In addition, both PS-NPs and PS-MPs increased Cu accumulation in D. magna on the CuO-NPs exposure. Specifically, under 0.1 mg L−1 CuO-NP exposure, PS-NPs and PS-MPs elevated Cu contents in D. magna by 34% and 127%, respectively, whereas under 1 mg L−1 CuO-NP exposure, the increases were 51% and 254%. Mechanistically, the co-exposure to CuO-NPs and PS-NPs disrupted Cu absorption, ion homeostasis, and autophagic processes in D. magna, whereas PS-MPs exacerbated CuO-NP toxicity primarily by interfering with oxidative phosphorylation pathways. The results underscore the elevated ecological risk posed by the co-occurrence of micro- and NPs and metal-based NPs in aquatic environments.