Background: 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 health-care associated pneumonia may enter the lungs. Limited evidence has been found implicating Acanthamoeba spp. as a primary cause of pneumonia and urinary catheter colonisation in intensive care patients. Aim: To explore the possibility of colonisation of the respiratory and urinary tracts of intensive care patients with free-living amoebae. Methods: Thirty-nine catheter urine, 50 endotracheal trap sputa and one general ward sputum sample from 45 patients and nine intensive care unit (ICU) environmental water samples were collected over a four and half month period in the Royal Hobart Hospital (RHH) from August 2011. Findings: Acanthamoebae were isolated by culture and detected by PCR 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. Conclusion: Colonisation of ICU patient’s 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 patient’s respiratory tracts.