Pseudomonas aeruginosais an opportunistic pathogen that infects patients by regulating virulence factors and biofilms through a quorum sensing (QS) system to protect itself from antibiotics and environmental stress. Therefore, the development of quorum sensing inhibitors (QSIs) is expected to become a new strategy for studying drug resistance toP. aeruginosainfections. Marine fungi are valuable resources for screening QSIs. A marine fungus,Penicilliumsp. JH1, with anti-QS activity was isolated from the offshore waters of Qingdao (China), and citrinin, a novel QSI, was purified from secondary metabolites of this fungus. Citrinin could significantly inhibit the production of violacein inChromobacterium violaceumCV12472 and the production of three virulence factors (elastase, rhamnolipid and pyocyanin) inP. aeruginosaPAO1. It could also inhibit the biofilm formation and motility of PAO1. In addition, citrinin downregulated the transcript levels of nine genes (lasI,rhlI,pqsA,lasR,rhlR,pqsR,lasB,rhlAandphzH) associated with QS. Molecular docking results showed that citrinin bound to PqsR and LasR with better affinity than the natural ligands. This study laid a foundation for the further study of the structure optimization and structure–activity relationship of citrinin.