With the development of antimicrobial agents, researchers have developed new strategies through key regulatory systems to block the expression of virulence genes without affecting bacterial growth. This strategy can minimize the selective pressure that leads to the emergence of resistance. Quorum sensing (QS) is an intercellular communication system that plays a key role in the regulation of bacterial virulence and biofilm formation. Studies have revealed that the QS system controls 4–6% of the total number ofP. aeruginosagenes, and quorum sensing inhibitors (QSIs) could be a promising target for developing new prevention and treatment strategies againstP. aeruginosainfection. In this study, four series of phenyloxadiazole and phenyltetrazole sulfoxide derivatives were synthesized and evaluated for their inhibitory effects onP. aeruginosaPAO1 biofilm formation. Our results showed that5bhad biofilm inhibitory activity and reduced the production of QS-regulated virulence factors inP. aeruginosa. In addition, silico molecular docking studies have shown that5bbinds to theP. aeruginosaQS receptor protein LasR through hydrogen bond interaction. Preliminary structure–activity relationship and docking studies show that5bhas broad application prospects as an anti-biofilm compound, and further research will be carried out in the future to solve the problem of microbial resistance.