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INTRODUCTION Bloodstream Infections (BSIs) are a main cause of life-threatening complications among patients with cancer. METHODOLOGY This study aimed to identify microbial pathogens causing BSI in febrile neutropenic patients with hematologic malignancy and compare the results of conventional blood culture with a nested multiplex real time PCR assay done directly on whole blood samples. The nested multiplex PCR was based on 16S rDNA and 18S rDNA sequence-specific primers; hence, it allowed the identification of most species of bacteria and fungi. RESULTS Forty adult patients with febrile neutropenia, admitted at Hematology ward of Ain Shams University Hospitals, were included in this study. Each patient was subjected to conventional blood culture and nested multiplex PCR. Blood culture was positive in 19 patients (47.5%). About 68.4% of the positive cultures were monomicrobial, while 31.6% were polymicrobial. A total number of 26 isolates were grown from positive cultures; Staphylococcus aureus was the most common (30.8%), followed by Klebsiella pneumoniae (19.2%). Regarding nested PCR, positive results were detected in 37/40 patients (92.5%) which was statistically significantly higher than that of blood culture. Eighteen samples that tested negative by culture were positive using the molecular approach. The agreement between the two approaches was 55%. CONCLUSION nested multiplex real time PCR can be a promising tool in order to achieve rapid diagnosis in cancer patients clinically suspected of BSIs. Its utilization could affect the choice of antimicrobial treatment whether bacterial or fungal and, therefore avoid unnecessary use of antimicrobials.