A study on the presence of free-living amoebae in a public hospital was developed in the city of Porto Alegre, State of Rio Grande do Sul. Dust and biofilms were collected using sterile swabs that had been prepared for this study, from 15 hospital environments, including the intensive care center, pediatric intensive care unit, kitchen, emergency room, outpatient surgical center, clinical surgical center, water storage tanks, taps and six drinking fountains for general use, every month from July 2004 to March 2005. The FLAs were isolated by culturing, using non-nutrient agar medium with the addition of heat-killed Escherichia coli. The protozoa were identified by morphological observation of cysts and trophozoites, in accordance with Page's morphological criteria (1988). Among the 135 samples collected from the 15 environments, 47 (35%) were positive for FLAs. Of these, thirty-four percent presented morphological characteristics particular to the genus Acanthamoeba.