Copper nanoparticles arise in soil and water from different industries, and well known for their adverse effects on aquatic and terrestrial ecosystems. The soil and hydroponic experiments were conducted to assess the toxicity of excessive amounts of CuO nanoparticles spiked in the soil and in hydroponic conditions on the root and leaf cells of spring barley. The microscopic study indicated changes in vascular bundles, stele, accumulation of electronically dense material in epidermis and vacuoles. The root hairs reduced by excess CuO nanoparticles even disappeared in hydroponic condition. Moreover, there were no significant changes visualized in semithin sections of leaf cells. The descriptive study on ultrastructure also indicated several changes in the chloroplast, thylakoids, plastoglobules, starch granules, peroxisomes and mitochondrial cristae. The negative effects of CuO nanoparticles on barley were more significant in the aquatic medium than in the soil.