Background. Invasive aspergillosis (IA) is associated with high mortality. Successful outcome with treatment is linked to early diagnosis. The utility of classic diagnostic methods, however, is limited. Methods. To aid in the diagnosis of IA, we retrospectively assessed our diagnostic service, using real-time polymerase chain reaction (PCR) and galactomannan sandwich enzyme-linked immunosorbent assay (ELISA). Results. A total of 203 patients at risk of invasive fungal infection were screened by PCR, and 116 of the patients were also tested by ELISA. The patient group comprised 176 patients with hematological malignancy and 28 control patients with evidence of invasive candidal infection. Consensus European Organisation for Research and Treatment of Cancer and Mycoses Study Group criteria were used to classify fungal infection, which, by definition, excluded the PCR result. The PCR method was sensitive (up to 92.3% sensitivity) and specific (up to 94.6% specificity) and had good agreement with the galactomannan ELISA (76.7%) and high-resolution computed tomography scan results. Conclusions. A negative PCR result can be used to rule out IA and to limit the need for empirical antifungal therapy; thus, it has a role in diagnosing IA infections, especially in combination with antigen testing. PCR-positive cases classified as "false positives" regularly reflect the limitations of classic microbiological procedures or restricted use of consensus clinical methods employed to classify infection.