Exposure to bioburden (comprising bacteria and fungi) has become one of the critical issues for occupational and public health approaches in hospital environment. This study aims to assess the fungal burden in one Central Hospital in Oporto, Portugal, applying the Indoor Air Quality (IAQ) Portuguese legislation as guidance, verifying both the limit values and the conformity established for general indoor environments. Air samples were collected using a microbiological air sampler in several hospital areas (Day hospital, Internment ward, Operating Room, and Outpatient area). Among the 15 rooms analyzed, 20% (3 out of 15) presented I/O > 1 and 4 out of 15 presented Aspergillus section Fumigati, in a concentration above 12 UFC·m−3. Besides Aspergillus section Fumigati, the sections Nigri, Aspergilli, Usti and Versicolores were also observed. Qualitative assessment, besides the quantitative, should always be performed to guarantee an accurate assessment, even though the quantitative cut-off complies with the legal requirement. A multi-approach on sampling methods and in assays (culture based and molecular methods) will enrich data findings, enabling exposure assessors to perform IAQ assessments.