Bioaerosols can alter the air quality, affecting differently people depending on their health status. Methods to sample bioaerosols are limited in terms of sampling time and air volumes. This study evaluated a new methodology to sample airborne microorganisms in buildings, using the air handling unit (AHU) filters and filtration media sampling discs (FMSD). Filtration media with FMSD of different natures were tested at the laboratory, and characterized by their air permeability, particle collection efficiency and the airflow distribution on their surface. Filtration media with FMSD of the same nature had best properties with a sampling particle efficiency of the FMSD of 50 %. This sampling methodology was applied on a building AHU, and FMSD were collected each month during one year. Airborne microorganisms collected by the FMSD were analyzed by culture and nucleic acids based methods (qPCR and Next-Generation sequencing). FMSD methodology gave results for all of those identification methods. Most of the bacteria orders identified by 16 S sequencing were Rhodobacterales (Paracoccus), Pseudomonadales and Cyanobacteria. For fungi, the most represented orders were Agaricales, Polyporales and Hymenochaetales. This sampling methodology has also made it possible to detect small size airborne microorganisms like viruses including two Coronaviruses. This study has shown the potential of using AHU filters to sample the airborne microorganisms in buildings.