Mushrooms have been accepted as nutraceutical foods because of their high nutritional and functional values. They have also gained interest due to their medicinal properties, economic importance, and organoleptic merit. In this study, wild Ganoderma lucidum and four commercial mushrooms, that is, Pleurotus ostreatus, Volvariella volvacea, Hericium erinaceus, and Lentinus edodes from Pakistan were screened for their biological activities such as anticancer, antityrosinase, anti‐α‐glucosidase, and antithrombotic activities from their methanol, ethanol, and water extracts. Enzyme inhibition assay showed that selected mushrooms are potent inhibitors with %age inhibition ranging from 19.00% to 80.91%, and 32.85% to 83.38% for tyrosinase and α‐glucosidase, respectively. The best tyrosinase inhibition was shown by P. ostreatus whereas L. edodes was found best as α‐glucosidase inhibitor. These mushrooms were tested against cancer cell lines including HT‐29 colon and H‐1299 lungs carcinoma cell lines. G. lucidum showed 29% and 24% viability of cells against HT‐29 and H‐1299 cell lines, respectively. This antiproliferative effect was in dose‐dependent manner, and the maximum inhibition was observed at 200 μg/ml. Mushrooms extracts were also found effective against clot lysis. The percentage of clot lysis was in the range of 27%–29%. The research would provide knowledge to the people of Pakistan about the importance of locally available commercial mushrooms and wild mushrooms for health improvement and prevention against different kinds of diseases.