We describe the molecular characterization of a multiresistant Pseudomonas aeruginosa clone causing an outbreak in the intensive care unit (ICU) of a tertiary-care university hospital. Analysis included antimicrobial susceptibility profile, O-serotyping, pulsed-field gel electrophoresis, and amplified fragment length polymorphism. Resistance mechanisms were characterized, including production of naturally occurring and acquired beta-lactamases, porin expression, and efflux pump systems. Eighteen patients were colonized or infected with multiresistant P. aeruginosa. Multiresistant P. aeruginosa was panresistant to penicillins, cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones and remained susceptible only to colistin. Sixteen isolates (89%) belonged to serotype O:11, pulsed-field gel electrophoresis type A1, and amplified fragment length polymorphism type A. Resistance characterization of this epidemic clone showed an overexpression of the chromosomal cephalosporinase AmpC combined with decreased expression of porin OprD and the absence of metallo-beta-lactamase or extended-spectrum beta-lactamase. An upregulation of the MexXY efflux system due to an agrZ mutation in the mexZ repressor was detected. This epidemic clone was restricted to the ICU and was not found elsewhere in hospital. Contamination of the ICU environment and the hands of an ICU nurse with this clone suggests possible hand-borne transmission. Implementation of contact precautions effectively controlled transmission of the epidemic clone. This study illustrates the ability of multiresistant P. aeruginosa to cause an outbreak with significant morbidity and mortality and underscores the need to identify clonal outbreaks, which require targeted infection control measures.