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Description: Abstract: Molecular methods have become integral to microbiological research for microbial iden-17 tification. This literature review focuses on the application of molecular methods in examining air-18 borne bacteria and fungi in healthcare facilities. In January 2024, a comprehensive electronic search 19 was carried out in esteemed databases including PubMed, Web of Science, and Scopus, employing 20 carefully selected keywords such as ((bacteria) OR (virus) OR (fungi)) AND (aerosol) AND ((hospi-21 tal) OR (healthcare) OR (dental office)) AND ((molecular) OR (PCR) OR (NGS) OR (RNA) OR 22 (DNA) OR (metagenomic) OR (microarray)), following the PRISMA protocol. The review specifi-23 cally targets healthcare environments with elevated concentrations of pathogenic bacteria. A total 24 of 487 articles were initially identified, but only 13 met the inclusion criteria and were included in 25 the review. The study disclosed that the prevalent molecular methodology for appraising aerosol 26 quality encompassed the utilization of the PCR method, incorporating either 16S rRNA (bacteria) 27 or 18S rRNA (fungi) amplification techniques. Notably, five diverse molecular techniques, specifi-28 cally PFGE, DGGE, SBT, LAMP and DNA hybridization methods, were implemented in five distinct 29 studies. These molecular tests exhibited superior capabilities compared to traditional bacterial and 30 fungal cultures, providing precise strain identification. Additionally, the molecular methods al-31 lowed the detection of gene sequences associated with antibiotic resistance. In conclusion, molecu-32 lar testing offers significant advantages over classical microbiological culture, providing more com-33 prehensive information.