The exposure to fungal burden is an increased risk factor for the development of respiratory symptoms and fungal diseases. The emergence worldwide of azole resistance in fungal species is a major concern for public health. Clinical settings must provide a clean and safe environment to protect patients and staff from fungal diseases. The aim of this study was to assess the fungal burden and prevalence of azole resistance in clinical settings in Portugal using passive sampling methods. Ten Primary Health Care Centers were evaluated through passive sampling, including electrostatic dust cloths, heating ventilation and air conditioning filters, and settled dust. All samples were plated onto Sabouraud dextrose agar media and screened for azole resistance using agar media supplemented with itraconazole, voriconazole and posaconazole through incubation at 27 °C, for 5 days. Fungal species were identified based on macro and micromorphology. Fungal load ranged from 348 to 424628 CFU·m−2 in EDC, 0 to 56500 CFU·m−2 in HVAC filter, and 2 to 514 CFU·g−1 in settled dust. EDC samples presented the highest fungal loads and wider diversity. Azole resistance was observed in all Primary Health Care Centers. Multi-azole resistance (fungal growth in two or more azoles) was observed in 90% units for Penicillium sp., C. sitophila, and Cladosporium sp. None of the samples containing Aspergillus sp. colonies presented resistance to the tested azoles. The knowledge of the fungal burden and prevalence of resistance to azole-based antifungal drugs in clinical environments will allow a better risk characterization regarding fungal burden.