Abstract: Background: Fungi are the good source of various biological active secondary metabolites. Materials and Methods: The antioxidant potential of Penicillium expansum isolated from soil of Punjab, India was studied and a three-step optimization strategy which includes, one-factor-at-a-time classical method and different statistical approaches (Plackett-Burman design and response surface methodology) were applied to enhance the antioxidant potential. Antioxidant activity was assayed by different procedures and compared with total phenolic content. Results: Primarily, different carbon and nitrogen sources were screened by classical methods, which revealed sucrose as carbon source is most suitable for antioxidant activity. Sodium nitrate, yeast extract and peptone were good sources of nitrogen but sodium nitrate was the best among them. Significance of the components of Czapek doxs medium with respect to antioxidant activity was evaluated with Plackett-Burman design, which supported sucrose and NaNO3 to be the most significant. In second step, sucrose and NaNO3 along with temperature were taken as three variables for response surface methodology to study their interaction. Response surface analysis showed significant enhancement in the antioxidant potential of Penicillium expansum. A compound was purified from the ethyl acetate extract which demonstrated potent antioxidant activity. Conclusion: The present study demonstrated potential of soil fungi to have antioxidant activity similar to plants and mushrooms thus further highlighting their significance as new sources of natural antioxidants and thus endorse the future prospects for the commercial production of natural and safer antioxidant compounds from such fungi. The fungi may provide easier set up for production and purification of natural antioxidants as compared to higher plants.