Cucurbita maxima, is a therapeutic plant spread all over the world. The seed of C. maxima constitutes a large amount of alkaloids, phenolic compounds, Vitamin E, and other secondary metabolites. The experiment was performed in four setups (Control, Only H2O2, H2O2 + 25 mg of alkaloid, H2O2 + 50 mg of alkaloids). The enzymatic and nonenzymatic antioxidants and lipid peroxidation (LPO) were determined to analyze the antioxidant effect of partially purified alkaloids. The results showed that partially purified alkaloids from C. maxima seed reacts effectively on the erythrocytes and leukocytes antioxidant enzyme system when compared to the hydrogen peroxide group. The present results indicate that the alkaloids present in this plant can be used as a natural antioxidant for the pharmacological purposes. Practical applications The C. maxima seed constitutes rich source of alkaloids, phenolic compounds, Vitamin E, and other secondary metabolites. The results obtained revealed that the purified alkaloids from C. maxima seed acts as natural antioxidants, which enhanced the potential to scavenge H2O2 and aids in protecting the RBC and WBC cells. The presence of 27 alkaloids of which seven major alkaloids possessing higher medicinal properties like antioxidant, antimicrobial, anti‐inflammatory, antitumor, anti‐aging, anti‐diabetic, anticancer, anti‐malarial, analgesic, nematicide, pesticide, and hemolytic activity were determined from the GC‐MS analysis of the extract. Alkaloids acts as major constituent in phytotheraphy and has wide range of clinical application in humans and animals. In conclusion, alkaloids from C. maxima seed may have several properties that leading to the opening of new avenues in the natural product for the therapeutic purpose.