<strong>BACKGROUND:</strong> Acanthamoebae are ubiquitous free-living environmental amoebae that may occasionally cause keratitis, granulomatous encephalitis, cutaneous lesions and systemic disease in humans. Acanthamoeba spp. have been implicated as a vehicle by which a number of common bacterial causes of healthcare-associated pneumonia may enter the lungs. Limited evidence has been found implicating Acanthamoeba spp. as a primary cause of pneumonia and urinary catheter colonization in intensive care patients.<p></p> <p><strong>AIM:</strong> To explore the possibility of colonization of the respiratory and urinary tracts of intensive care patients with free-living amoebae.</p> <p><strong>METHODS:</strong> Thirty-nine catheter urines, 50 endotracheal trap sputa and one general ward sputum sample from 45 patients and nine intensive care unit (ICU) environmental water samples were collected during a four-and-half-month period in the Royal Hobart Hospital from August 2011.</p> <p><strong>FINDINGS:</strong> Acanthamoebae were isolated by culture and detected by polymerase chain reaction in two sputum samples from a single patient, taken one week apart. A single Acanthamoeba species isolate was detected by culture only from the ICU environment.</p> <p><strong>CONCLUSION:</strong> Colonization of ICU patients' respiratory tracts with Acanthamoeba spp. does occur. This may have significance for the role of acanthamoebae as a source of bacterial pathogens in intensive therapy patients' respiratory tracts.</p>