Pseudomonas aeruginosa, an opportunistic pathogen, is the third most common pathogen associated with nosocomial urinary tract infections (UTIs). The virulence of this organism is due to its ability to produce quorum-sensing (QS) signal molecules and form biofilms. These biofilms are usually resistant to conventional antibiotics and host immune responses. Recently, beneficial effects of macrolides, especially azithromycin (AZM), have been shown in patients suffering from chronic infections caused by P. aeruginosa. These were due to anti-inflammatory and modulatory effects of AZM on the expression of virulence factors of this pathogen. The present study was designed to evaluate the potential of AZM to inhibit QS signal molecules and its ability to attenuate the virulence of P. aeruginosa in an experimental UTI model. Sub-MIC concentrations of AZM significantly inhibited the production of QS signals, swimming, swarming and twitching motilities, and biofilm formation in vitro. The therapeutic evaluation of AZM in this experimental UTI model showed complete clearance of the organisms from the mouse kidneys. The results of this study highlight the potential effectiveness of AZM in attenuating the virulence of P. aeruginosa in a UTI model.