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The effects of copper nanoparticles (Cu-NPs), including their stability in the medium, were studied with the green unicellular algae Chlamydomonas reinhardtii (CC-125). Cu-NPs were synthesized and characterized. Cu-NP particles were uniform, regular, and largely spherical, and they had smooth surfaces; the average size was estimated to be 137.4 ± 2.1 nm. Chlamydomonas cells were cultivated for 96 h under controlled conditions in the presence of Cu-NPs, according to OECD guidelines, and then subjected to toxicological bioassays. Based on scanning electron microscopy (SEM) observations, the effects of Cu-NPs resulted in part from the dissolution of nanoparticles (NPs) and the action of copper itself, which shows the importance of studying NP stability in the testing environment. In this assay, deleterious effects were enhanced by increasing Cu-NP concentrations (5, 10, 25, 50, and 100 mg/L). Concentrations higher than 25 mg/L exhibited extreme toxicity. We confirmed the known toxic effects of metal NPs, namely, growth inhibition, reduction of chlorophyll levels in cells, cell penetration and increased ROS production. Attention was also paid to select underexplored metabolites, which were studied with a LC-MS/MS system. Treatments caused changes in metabolites profiles, and levels of p-hydroxybenzaldehyde and protocatechuic acid were especially enhanced, suggesting their positive roles in the antioxidant defence response. Furthermore, a repeatable increase in suberic acid levels was observed for various stress conditions tested, and we expect that this was the result of lipid peroxidation.