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The objective of this study was to evaluate the composition of bioactive compounds and the antioxidant activity of an Algerian variety of virgin olive oil (VOO) called “Rougette of Mitidja,” produced in a desert region, as well as its protective effects against oxidative stress induced by titanium dioxide nanoparticles (NPTiO2). The bioactive compounds were analyzed using UV-visible spectrophotometry, while antioxidant activity was tested in vitro using DPPH and in vivo in rats. A total of 24 male albino Wistar rats were randomly divided into 4 groups: control, NPTiO2, NPTiO2 + VOO1, and NPTiO2 + VOO2. The analysis revealed that the VOO contained polyphenols, flavonoids, and carotenoids with significant DDPH antioxidant activity at 87.70%. NPTiO2 induced oxidative stress by increasing biochemical parameters and hepatic and renal lipid peroxidation, as evidenced by a rise in MDA with a decrease in GSH and antioxidant enzyme activities in rats. NPTiO2 also affected the hematological profile, leading to a decrease in red blood cells and hemoglobin. Treatment with VOO exerted a protective effect by lowering biochemical parameters and increasing antioxidant enzymes compared to the NPTiO2 group. Histological findings confirmed the protective effects of VOO on liver and kidney damage caused by NPTiO2. It can be concluded that monovarietal VOO has a potent protective effect against oxidative stress induced by NPTiO2, which is related to its richness in bioactive compounds.