<p>The extensive commercial use of copper oxide nanoparticles (CuO NPs) paves their way to agri-environment. <i>Ocimum basilicum</i> exhibits medicinal potential and commercially cultivated for its essential oil (EO) globally. The present study focused on investigating the differential uptake of CuO NPs/Cu⁺⁺ (0, 0.2, 0.4&#xa0;µg ml⁻¹) and their possible adverse impact on <i>O. basilicum.</i> The transmission électron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS) analyses of exposed plant tissues clearly reflect the uptake, translocation and subcellular distribution of CuO NPs by apoplastic and symplastic (plasmodesmata) pathways. The ultra-structural adverse effects of CuO NPs include swelling of endoplasmic reticulum, dissolution of cristae, grana and cellular membrane damage. CuO NPs and Cu⁺⁺ exhibit distortion in stomatal morphology, reduction in whole plant biomass (CuO NPs: 36.93%; Cu⁺⁺: 53.86%) and photosynthetic pigments at 0.4&#xa0;µg ml⁻¹ CuO NPs/ Cu⁺⁺ exposure. Further, the concentration dependent increase in oxidative stress markers (malondialdehyde and hydrogen peroxide) has been evident in both root and leaves of the exposed plants. This study also documented the significant upregulation of cellular and enzymatic antioxidants (superoxide dismutase and catalase). However, guaiacol peroxidase enzyme activity was significantly down regulated (maximum decrease of 47.98% and 73.83% in leaves at 0.4&#xa0;µg ml⁻¹ CuO NPs and Cu⁺⁺ respectively) compared to untreated control plants. Major EO constituents of <i>O. basilicum</i> (caryophyllene, germacrene D, eugenol, ocimene, methyl-chavicol, geraniol and nerol) were also affected by CuO NPs exposure. The findings of this study, highlighted the phytotoxic risks associated with the CuO NPs and emphasizing the need to regulate the CuO NPs use in agriculture and commercial products for safe and sustainable environment.</p>