The relationship between air contamination (cfu/m(3)) with fungal spores, especially Aspergillus spp., in three renovation areas of a neonatal intensive care unit (NICU) and colonization and infection rates in a high care area (HC) equipped with high efficiency particulate air (HEPA) filtration and a high pressure system, was evaluated. Data on the type and site of renovation works, outdoor meteorological conditions, patient crowding and nasopharyngeal colonization rate were collected. Factors not associated with Aspergillus spp. concentration were outdoor temperature, air pressure, wind speed, humidity, rainfall, patient density in the NICU, renovation works in the administrative area and in the isolation rooms. Multivariate analysis revealed that renovation works and air concentration of Aspergillus spp. spores in the medium care area (MC) resulted in a significant increase of the concentration in the HC of the NICU. The use of a mobile HEPA air filtration system (MedicCleanAir(R)Forte, Willebroek, Belgium) caused a significant decrease in the Aspergillus spp. concentration. There was no relationship between Aspergillus spp. air concentration and nasopharyngeal colonization in the neonates. Invasive aspergillosis did not occur during the renovation. This study highlights the importance of optimal physical barriers and air filtration to decrease airborne fungal spores in high-risk units during renovation works. The value of patient surveillance and environmental air sampling is questionable since no relationship was found between air contamination and colonization in patients.