Pseudomonas aeruginosa biofilm lifestyle exhibits multidrug resistance in chronic bacterial infections. Alternative antimicrobial compounds or combination drug therapies must be urgently developed. In this work, the antibiofilm effect of Ag nanoparticle (AgNP) combined with the quorum sensing inhibitor (QSI) 4-nitropyridine N-oxide (4NPO) on P. aeruginosa biofilms was investigated. The biofilm biomass of P. aeruginosa was considerably reduced by 1.56-50mg/L AgNP. However, 4NPO enhanced the ability of AgNP to inhibit P. aeruginosa biofilm formation (P<0.05). The combination of AgNP with 4NPO could continuously inhibit biofilm development after 12h, and 50mg/L AgNP combined with 6.25mg/L 4NPO thoroughly suppressed biofilm growth. The expression levels of QS genes and exopolysaccharide genes of biofilm treated with the combination of AgNP with 4NPO (AgNP-4NPO combination) were lower than those treated with AgNP alone (P<0.05). Additional extracellular proteins and polysaccharides were determined in the samples treated with AgNP-4NPO combination. Based on proteomic analysis, this result was attributed to cell rupture caused by antimicrobial agents and intracellular materials released. The combination of the two antimicrobial agents could weaken the swimming ability of bacterial cells by damaging bacterial flagella and blocking rhlA gene expression. Thus, AgNP combined with QSI showed stronger antibiofilm ability than AgNP alone. These results may contribute to the development of antimicrobial agents.