Brown-colored copper oxide nanoparticles (CuO NPs) were synthesized using a green synthesis method and characterized using X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FT-IR). These nanoparticles have gained significant interest during the last decade in various fields owing to their unique physical and chemical properties. However, there have been concerns regarding their toxicological potential and possible adverse health effects on living organisms and the environment. Hence, this study was conducted to unveil the mechanisms of toxicity of copper oxide nanoparticles (CuO NPs) on vital organs (brain, heart, testes, and muscles) of albino rats. After acclimatization, the rats in groups G-G were exposed to CuO NPs via intraperitoneal route at 250, 500, and 750 mg/kg/day, respectively, for 20 days. Different behavioral changes, oxidative stress biomarkers, and histopathological alterations were recorded. Results revealed a significant dose-dependent increase in the contents of reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) and a reduction of antioxidant enzymes (CAT, POD, and SOD) in different visceral organs. At the histopathological level, the brain of treated rats showed neuronal atrophy, degeneration of neurons, necrosis, and inflammatory reactions. Various sections of the testes of rats at high doses exhibited tubular degeneration, sloughing/necrosis of epithelium, and arresting of spermatogenesis, while heart sections indicated congestion, inflammatory exudate, myofibrillolysis, neutrophilic myocarditis, and coagulative necrosis. From the findings of this study, it is recorded that long-term exposure to CuO NPs induces toxicological impacts via induction of oxidative stress and disruption of physiological functions of multiple tissues of albino rats.