Agrocybe aegerita (Brig.) Sing is a basidiomycete, white rot fungus. Antimicrobial activities and the antiqourum effect on Pseudomonas aeruginosa of an A. aegerita methanolic extract were investigated. The extract showed very good antimicrobial activity against all the tested microorganisms in a dose dependent manner. Effects of the Sub-MIC, MIC and 2MIC of the A. aegerita methanolic extract regulated the virulence factors in the quorum sensing (QS) test, as well as biofilm formation on P. aeruginosa. Sub-inhibitory and inhibitory concentrations of the extract demonstrated the reduction of virulence factors such as pyocyanin production, twitching and swimming motility. The biofilm forming capability of P. aeruginosa PAO1 was also reduced in a concentration-dependent manner. Furthermore, the chemical composition of the methanolic extract was determined considering its phenolic composition. The methanolic extract of A. aegerita can be a very good source of bioactive substances. This research is of great importance due to the prevalence of drug-resistant microorganisms.