The rapid diagnosis of pathogens and prompt initiation of appropriate antibiotic therapy are critical factors to reduce the morbidity and mortality associated with sepsis. In this study, we evaluated a multiplex polymerase chain reaction (PCR-M) test that detects bacteria and fungi in whole-blood specimens, comparing its features to those of a blood culture (BC). Following evaluation of the performance for sensitivity and specificity of PCR-M, 78 blood samples from 54 patients with suspected bacterial infections were evaluated. Whole-blood samples for PCR-M were collected at the same time as BC, and PCR-M results were compared with BC results. As a result, minimum sensitivity of the kit was 1-100 cfu/ml. The PCR-M test correctly identified specificity for 13 out of 14 strains blinded to the assay analyst. Of 78 blood samples examined, 56 (72%) were negative by both methods, and 22 (28%) were positive by at least one of the two methods. PCR-M detected organisms in 21 cases (27%) compared with 12 cases (15%) in BC. The correlation of positives between PCR-M and BC was 92% (11/12), and both methods identified the same organisms in these 11 cases. With higher positive rate compared with BC, PCR-M could detect and identify potentially significant microorganisms within a few hours by using a small volume of a single whole-blood sample. Early detection of microorganisms has the potential to facilitate early determination of appropriate treatment and antimicrobial selection.