Abstract: Background and Objective: There is an increasing interest to replace chemical preservatives by natural antioxidants from essential oils of plants in food industry. This study was designed to determine the optimal conditions leading to the maximum extraction yield of essential oil of Mentha pulegium L. collected from Algeria by ultrasound-assisted hydrodistillation, using response surface methodology (RSM) and to evaluate its antioxidant activity. Materials and Methods: The effects of three process parameters on extraction yield have been studied using the Box-Benken design. The antioxidant activity of essential oil was investigated through 2 methods, scavenging capacity of DPPH and reducing power. Analysis of variance (n-way ANOVA) used for statistical analysis of the model was evaluated by Fisher test (F-value). Results: Analysis of variance (n-way ANOVA) revealed that the model was significant (p<0.0001). The optimum extraction yield (2.42%) was obtained at an extraction time of 60 min, an ultrasound power of 60 W and a plant material to water ratio of 1:12 g mL–1. The essential oil of Mentha pulegium L. was found to have an appreciable antioxidant potential against DPPH (IC50 = 1210.11 mg L–1) and reducing power. Conclusion: The response surface methodology was an appropriate technique for optimization of extraction yield of Mentha pulegium L. essential oil by ultrasound-assisted hydrodistillation. The results of antioxidant activity suggest that the essential oil have a huge potential as an alternatives to synthetic preservatives in food industry.