This study analyzed 57 Aspergillus section Fumigati (AF) isolates collected by active and passive sampling (N = 450) in several health care facilities and from biological sampling of health care workers (N = 25) and controls (N = 22) in Portugal. All isolates were cultured in different media and screened for azole resistance. Cytotoxicity was assessed for 40 isolates in lung epithelial cells and kidney cells using the MTT assay. Aspergillus section Fumigati was prevalent in the health care facilities and in nasal swabs from health care workers and controls. All AF isolates reduced cell viability and presented medium to high cytotoxicity, with cytotoxicity being significantly higher in A549 lung epithelial cells. The cytotoxicity of isolates from air and nasal swab samples suggested the inhalation route as a risk factor. Notably, 42% of AF isolates exhibited a pattern of reduced susceptibility to some of the most used antifungals available for the treatment of patients infected with these fungi. In sum, the epidemiology and clinical relevance of Aspergillus section Fumigati should continue to be addressed. A deeper understanding of the mechanisms underlying Aspergillus-mediated cytotoxicity is necessary.