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This work aimed to study the toxic implications of zinc oxide nanoparticles (ZnO NPs) on the physio-biochemical responses of spring barley (<i>Hordeum sativum</i> L.). The experiments were designed in a hydroponic system, and <i>H. sativum</i> was treated with two concentrations of ZnO NPs, namely 300 and 2000 mg/L. The findings demonstrated that ZnO NPs prevent the growth of <i>H. sativum</i> through the modulation of the degree of oxidative stress and the metabolism of antioxidant enzymes. The results showed increased malondialdehyde (MDA) by 1.17- and 1.69-fold, proline by 1.03- and 1.09-fold, and catalase (CAT) by 1.4- and 1.6-fold in shoots for ZnO NPs at 300 and 2000 mg/L, respectively. The activity of superoxide dismutase (SOD) increased by 2 and 3.3 times, ascorbate peroxidase (APOX) by 1.2 and 1.3 times, glutathione-s-transferase (GST) by 1.2 and 2.5 times, and glutathione reductase (GR) by 1.8 and 1.3 times in roots at 300 and 2000 mg/L, respectively. However, the level of δ-aminolevulinic acid (ALA) decreased by 1.4 and 1.3 times in roots and by 1.1 times in both treatments (nano-300 and nano-2000), respectively, indicating changes in the chlorophyll metabolic pathway. The outcomes can be utilized to create a plan of action for plants to withstand the stress brought on by the presence of NPs.