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Many of the virulence factors produced by the opportunistic human pathogen Pseudomonas aeruginosa are quorum‐sensing (QS) regulated. Among these are rhamnolipids, which have been shown to cause lysis of several cellular components of the human immune system, e.g. monocyte‐derived macrophages and polymorphonuclear leukocytes (PMNs). We have previously shown that rhamnolipids produced by P. aeruginosa cause necrotic death of PMNs in vitro. This raises the possibility that rhamnolipids may function as a ‘biofilm shield’in vivo, which contributes significantly to the increased tolerance of P. aeruginosa biofilms to PMNs. In the present study, we demonstrate the importance of the production of rhamnolipids in the establishment and persistence of P. aeruginosa infections, using an in vitro biofilm system, an intraperitoneal foreign‐body model and a pulmonary model of P. aeruginosa infections in …