Nanoparticle (NP) pollution is a worldwide problem. Copper oxide nanoparticles (CuO NPs) are one of the most used NPs in a variety of applications, which results in their increased release into the marine environment. In the present work, the marine mussel Lithophaga lithophaga was used as a model organism to evaluate the toxic effects of CuO NPs following 28 days of exposure to sub-lethal concentrations (5 and 20 μg/L). The time points were 1 day of exposure to assess the cell viability, phagocytosis in mussel haemocytes and genotoxicity (DNA damage in gills), 1, 14 and 28 days of exposure to evaluate copper concentrations in water and gills, as well as metallothionein concentration in gills, while gill histology and SEM examination were done after 28 days of exposure. The results indicated that the accumulation of CuO NPs in gills increased with concentration and time. Mussel exposure to CuO NPs increased neutral red uptake. However, the phagocytic abilities decreased in haemocytes with increased concentration. CuO NPs caused DNA damage in the gills even at low concentrations (5 µg/L). CuO NPs caused histopathological alterations in gills, such as brown cell accumulation, necrosis, dwarfism of filaments and ciliary erosion. In conclusion, exposure of the mussel L. lithophaga to CuO NPs led to concentration- and time-dependent responses for all the examined biomarkers. Thus, L. lithophaga may be used as a bioindicator organism in the assessment of CuO NP toxicity.