Febrile neutropenia is the main treatment-related cause of mortality in cancer patients. During June 2012 to April 2013, 76 blood culture samples from patients receiving chemotherapy for hematological malignancy and cancer with febrile neutropenia episodes (FNEs) were examined for the presence of bacteria and fungi based on 16S rRNA gene and 18S rRNA combined with real-time PCR amplification and sequencing. Furthermore, we used a loopmediatedisothermal amplification (LAMP) assay for the detection of herpes simplex virus type 1 and 2 (HSV-1,2), varicella zoster virus (VZV), epstein-barr virus (EBV), cytomegalovirus (CMV) and human herpes virus type6 and 7 (HHV-6,7), followed by a real-time PCR amplification assay. Of these samples, bacteria were identified in 19 of 76 FNEs (25.0%) by a real-time PCR assay and in 9 of 76 (11.8%) by blood culture. In 6 blood culture-positive samples, real-timePCR assay detected the same type of bacteria. No fungus was detected both real-time PCR assay and blood culture. Viruses were identified in 6 of 76 FNE (7.9%). During antibiotic therapy, all samples were negative in blood culture, but a real time PCR assay yielded a positive result in 2 cases of 2 (100%). The number of bacteria DNA copy and serum CRP titer of patients with FNE did not correlated well. We conclude a real-time PCR assay could be given higher microbe's detection rate, and need shorter turnaround time, and smaller blood sample than blood culture. Using a real-time PCR assay combined with blood culture improves microbiological documentation in febrile neuropenia episodes.