Febrile neutropenia (FN) is a life-threatening complication, and the primary cause of FN is considered to be microbial infection. Therefore, prompt and appropriate antimicrobial therapy is crucial. Clinicians usually prescribe antimicrobial therapy on the basis of presumptive and empirical data. This is because the causative pathogen for FN in blood culture (BC) analysis is detected several days after sampling. Polymerase chain reaction (PCR) analysis has been used for detecting the causative bacteria of infections. Here, we examined whether multiplex PCR is useful for detecting the causative pathogens for FN patients. We extracted DNA from the patients' whole blood and performed multiplex PCR. In total, 128 samples of 40 patients clinically diagnosed with FN were used in this study. Multiplex PCR analysis revealed the causative pathogen in 3 patients with FN; the DNA fragments amplified were those of Pseudomonas aeruginosa in 2 cases and Psedomonas putida in 1 case. These patients could be started on appropriate antimicrobial therapy a few hours after sampling. However, the DNA fragment of the causative pathogen could not be amplified by PCR in 2 patients, although BC analysis did detect the causative bacteria. Thus, we conclude that multiplex PCR is serviceable in case of FN because of its rapidness. However, BC is also indispensable to treating FN owing to its high sensitivity.