Pseudomonas aeruginosa is a Gram-negative, rod-shaped bacterium responsible for severe and persistent infections in immunocompromised and cystic fibrosis patients. It is a highly recalcitrant pathogen because it is resistant to most antimicrobial agents, making infections difficult to treat and contain. This problem is further compounded by the ability of P. aeruginosa to produce a biofilm matrix, which increases antibiotic resistance by lowering the accessibility of antimicrobial agents to the bacterial cells. Quorum sensing (QS), a bacterial cell–cell communication and gene regulatory system that modulates the expression of virulence and biofilm formation genes, is a potential target for developing new therapies against P. aeruginosa infection. This chapter will focus on recent advances in targeting the four main QS systems (las, rhl, pqs, and iqs) of P. aeruginosa and will specifically evaluate their role in biofilm development.