To evaluate fungal microbiota in air-conditioning units installed in intensive care units in two university hospitals in Cuiaba city, Mato Grosso, central western region of Brazil, 525 solid environmental samples were collected, 285 from Hospital A and 240 from Hospital B. Collections were performed using sterile swabs on air-conditioning unit components: cooling coils, ventilators, and filters. Mycelial fungi identification was achieved by observation of the macroscopic and micromorphological characteristics in different culture mediums (maize meal, oatmeal and potato dextrose agars and malt extract) using the Ridell technique. Eleven genera and 27 distinct species belonging to the hyphomycetes and ascomycetes classes were isolated and identified. The most frequently detected genera in both hospitals were Aspergillus spp, Penicillium spp, and Cladosporium spp. Values for colony-forming units per gram were 64 and 75%, well above the limits recommended by Health Ministry resolution 176/00 at the locations selected for analysis in Hospitals A and B, respectively. In conclusion, evaluation of fungal microbiota in the air-conditioning units indirectly determined that the air quality was compromised in both university hospitals analyzed, which constitutes a risk factor for the acquisition of infection in the intensive care units.