To screen for the quorum-sensing (QS) inhibitors from marine-derived fungi and evaluate their anti-QS properties in Pseudomonas aeruginosa. QS inhibitory activity was found in secondary metabolites of a marine fungus Fusarium sp. Z10 using P. aeruginosa QSIS-lasI biosensor. The major active compound of this fungus was isolated by HPLC and identified as equisetin. Subinhibitory concentration of equisetin could inhibit the formation of biofilm, swarming motility, and the production of virulence factors in P. aeruginosa. The inhibition of las, PQS, and rhl system by equisetin were determined using Escherichia coli MG4/pKDT17, E.coli pEAL08-2, and E.coli pDSY, respectively. Real-time RT-PCR assays showed that equisetin could downregulate the mRNA expression of QS-related genes. Equisetin proved its potential as an inhibitor against P. aeruginosa QS system and might also serve as precursor compound in development of novel therapeutics for infectious diseases by optimal design of structures.