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OBJECTIVES This study aimed to evaluate the diagnostic usefulness of real-time (RT) polymerase chain reaction (PCR) on blood samples for diagnosis of invasive aspergillosis and mucormycosis in patients with suspected invasive mould infection. METHODS Adult patients with suspected invasive mould infection were prospectively enrolled at a tertiary referral hospital in Seoul, South Korea between 2017 and 2020. Standard tests for diagnosis of invasive mould infection and RT-PCR for Aspergillus, Mucor, and Rhizopus using blood samples were performed. We evaluated the diagnostic performance of RT-PCR tests in patients diagnosed with proven and probable invasive aspergillosis or mucormycosis infection, according to the modified definitions of the EORTC/MSG 2019. RESULTS A total of 102 patients with suspected invasive mould infection were enrolled. Of these patients, 46 (45%) were classified as having proven (n=13) or probable (n=33) invasive aspergillosis, 21 (21%) as proven (n=17) or probable (n=4) invasive mucormycosis, and 18 (18%) as possible invasive mould infection. The remaining 13 (13%) were classified as not having invasive mould infection. Patients with possible invasive mould infection (n=18) and coinfection of aspergillosis and mucormycosis (n=4) were excluded from the final analysis. The sensitivity and specificity of the Aspergillus PCR were 54.3% ([25/46], 95% confidence interval [CI]: 40.2-67.9%) and 94.1% ([32/34], 95% CI: 80.9-98.4%), respectively. The sensitivity and specificity of the Mucor or Rhizopus PCR were 57.1% ([12/21], 95% CI: 36.6-75.5%) and 76.3% ([45/59], 95% CI: 64.0-85.3), respectively. CONCLUSIONS Our study suggests that blood PCR can be a useful adjunct test for diagnosing patients with suspected invasive mould infection.