Background: Invasive fungal infection (IFI) is a fatal infection in haematology patients. There is an urgent need for reliable screening methods facilitating timely diagnosis and treatment. A real-time panfungal polymerase chain reaction (PCR) assay based on TaqMan technology targeting 18S ribosomal RNA gene was used to screen whole blood specimen obtained from series of Haematology malignancy patients for IFIs. Materials and Methods: The panfungal (Pan-ACF) assay was employed to investigate specimen from 133 patients in duplicate with suspected IFI. In addition twenty healthy subjects and twenty patients with bacterial infections were taken as control. The patients with suspected IFI were also diagnosed by conventional methods including direct microscopy, culture techniques and antigen detection (galactomannan antigen ELISA and latex agglutination for cryptococcal antigen). The results of molecular testing were evaluated in relation to the criteria proposed by the European Organization for Research and Treatment of Cancer and patients were classified as having proven and probable IFD. Results: Of 133 patients, 89 had proven, 18 had probable and 26 had possible IFI. One hundred four samples were reverse transcription-PCR positive. Of 89 proven cases, 84 were panfungal PCR positive. These 84 cases included 82 cases which revealed growth on fungal blood culture and two cases were negative on fungal blood culture. Of the 82 cases which revealed growth on culture: 74 grew Candida in culture, 3 grew Fusarium solani, 5 grew Aspergillus species on blood culture. The later five were also galactomannan antigen positive. The five specimen which were negative on panfungal PCR, two grew Trichosporon asahii, one grew Candida rugosa and two grew as Cryptococcus neoformans var. neoformans. Of the 18 probable cases, 18 were panfungal PCR positive. These were also galactomannan antigen positive. The sensitivity and specificity of panfungal PCR in proven cases were 94.3% and 95.2%, respectively. The positive and negative predictive values proven cases were 97.6% and 88.9%, respectively. Conclusions: The panfungal (Pan-ACF) real-time PCR assay can detect common fungal genera and it may be used as an adjunct to conventional methods for screening of IFI.