Background: This study provides an insight into the impact of ultrasound-assisted extraction with water as solvent (UAEW) and extraction by supercritical carbon dioxide (SC-CO2) with 5% EtOH on antioxidant and enzyme inhibitory activity in regard to the chemical profile of the edible and medicinal mushroom, Pleurotus pulmonarius. Results: Extraction efficiency was between 0.36% and 63.32%, depending on the extraction technique. The main compounds in the extracts were fatty acids. Supercritical CO2 extraction with co-solvent was the most suitable method for obtaining extracts that were rich in ergosterol content, reaching a value of 40.1 mg g−1. The UAEW of crude mushroom powder ensured the highest yield, as well as the extracts with best antioxidative activity. The measurements of enzyme inhibitory activity revealed that all types of investigated extracts exhibited only tyrosinase and amylase inhibition at a significant level. Conclusion: Based on our results, the extraction methods significantly affected the chemical profile and bioactivity of P. pulmonarius. © 2020 Society of Chemical Industry.