Copper nanoparticles demonstrate antibacterial activity, but their toxicity to eukaryotic systems is less understood. Here, we carried out a comparative study to determine the biocompatibility and cytotoxicity of sub-10 nm copper nanoparticles to a variety of biological systems, including prokaryotic cells (Escherichia coli), yeast, mammalian cell lines (HEK293T, PC12), and zebrafish embryos. We determined the bearing threshold for the cell-death-inducing concentration of copper nanoparticles by probing cell growth, viability, as well as embryological features. To exclude the partial toxicity effect from the remnant reactants, we developed a purification approach using agarose gel electrophoresis. Purified CuONP solution inhibits bacterial growth and causes eukaryotic cell death at 170 and 122.5 ppm (w/w) during the 18 h of treatment, respectively. CuONP significantly reduces the pigmentation of retina pigmented epithelium of zebrafish embryos at 85 ppm. The cytotoxicity of CuONP in eukaryotic cells could arise from the oxidative stress induced by CuONP. This result suggests that small copper nanoparticles exert cytotoxicity in both prokaryotic and eukaryotic systems, and therefore, caution should be used to avoid direct contact of copper nanoparticles to human tissues considering the potential use of copper nanoparticles in the clinical setting.