Nucleic acid-based assays have good potential to complement and enhance the sensitivity and rapidity of conventional methods used in diagnostic mycology. The majority of molecular tests are polymerase chain reaction (PCR)-based assays focusing mainly on the detection of Candida and Aspergillus spp. from clinical samples. DNA extraction and purification procedures should be standardized and can be facilitated by using commercial extraction kits. In general, protocols that target multi-copy genes provide the greatest sensitivity. Objective endpoint assessments of PCR tests using enzyme-linked immunosorbent assays (ELISA) or commercial quantitative systems are capable of rapidly detecting and identifying Candida and Aspergillus spp. Sequencing of PCR products can be used to confirm the identity of amplicons. In cases of suspected invasive aspergillosis, PCR should be performed on both blood and bronchoalveolar lavage fluid to maximize test sensitivity and the positive predictive value. At least two blood specimens should be tested if PCR is undertaken on blood samples alone. In situ hybridization techniques have been used with success to identify fungi in tissue specimens. The wide application of PCR-based assays relies on the introduction of standardized protocols following their evaluation in multicentre, prospective studies.