The objective of this study was to evaluate the antioxidant activity of different concentrations (250, 500 and 750 ppm) of Ocimum sanctum essential oil incorporated into soybean oil. The effect of O. sanctum essential oil at 250 ppm was similar to that of BHA on inhibiting oxidation in the soybean oil, but higher concentrations of essential oil (500 and 750 ppm) showed higher protection against oxidation in comparison with that of BHA. The influence of O. sanctum essential oil on stabilizing the soybean oil was more profound at 45 °C in comparison with 60 °C. In addition, the O. sanctum essential oil was significantly effective in protecting polyunsaturated fatty acids of soybean oil during storage at both temperatures of 45 and 60 °C. Generally, the O. sanctum essential oil can be introduced as a suitable natural antioxidant that can replace synthetic antioxidants.