Our body defends itself from oxidative stress via endogenous antioxidants. However, in case of deficiency, antioxidant supplements from plant and animal sources may help in maintaining overall health. The current research was conducted to evaluate the activity of enzymatic antioxidants like Superoxide dismutase, Ascorbate Peroxidase, Catalase, Glutathione Peroxidase, Glutathione Reductase, and non-enzymatic antioxidant Ascorbic acid in floret and stem of cauliflower and broccoli cultivar. Native PAGE was performed for SOD, CAT, and Glutathione Peroxidase. Antioxidant activity significantly varied among broccoli and cauliflower and their different parts. The activity of APX, GPX, and Protein was found maximum in Broccoli floret. Whereas SOD and CAT activity was higher in broccoli stems. Cauliflower floret had maximum ascorbic acid followed by broccoli floret. GR activity was highest in the cauliflower stem. Except for CAT, all other enzymatic antioxidant activity like SOD, APX, GPX, and GR was 2.5%, 47.59%,63.59%, and 23.6% higher in broccoli floret as compared to cauliflower floret. CAT activity was 29.64% higher in cauliflower floret compared to broccoli floret. Our findings support future efforts to utilize broccoli and cauliflower agro-waste as sources of compounds for the nutraceutical and pharmaceutical industries.