Agrocybe aegerita (Brig.) Sing is known as a white rot fungus. Antioxidant, antimicrobial activity and antiqourum effect on Pseudomonas aeruginosa of A. aegerita methanolic extract was investigated. The extract showed good antimicrobial activity. Effects of A. aegerita methanolic extract regulated virulence factors in quorum sensing (QS) test, as well as biofilm formation on P. aeruginosa and demonstrated reduction of pyocyanin production, twitching and swimming motility. Methanolic extract exhibited high antioxidant effect. The studied mushroom is a rich source of carbohydrates and proteins. Trehalose was the main free sugar while malic acid was the most abundant organic acid. α-, β-, γ-and δ-Tocopherols were also found, and oleic acid was the most prevalent fatty acid. A. aegerita can be a good source of nutritional and bioactive substances. This research is of great importance due to the prevalence of drug-resistant microorganisms and health-beneficial effects of antioxidants.