OBJECTIVES: New molecular methods allow rapid pathogen detection in patients with sepsis, but their impact on treatment decisions remains to be established. We evaluated the therapeutic usefulness of multiplex PCR testing in patients with cancer and sepsis. METHODS: 110 patients with cancer and sepsis were included prospectively and underwent LightCycler((R)) SeptiFast (LC-SF) multiplex PCR testing in addition to standard tests. Two independent panels of experts assessed the diagnosis in each patient based on medical record data; only one panel had the LC-SF results. The final diagnosis established by a third panel was the reference standard. RESULTS: The final diagnosis was documented sepsis in 50 patients (55 microorganisms), undocumented sepsis in 54, and non-infectious disease in 6. LC-SF detected 17/32 pathogens recovered from blood cultures (BC) and 11/23 pathogens not recovered from BC; 12 microorganisms were detected neither by BC nor by LC-SF. LC-SF produced false-positive results in 10 cases. The LC-SF results would have significantly improved treatment in 11 (10%) patients and prompted immediate antimicrobial therapy not given initially in 3 patients. CONCLUSIONS: In cancer patients with suspected sepsis, LC-SF detected 11/55 (20%) true pathogens not recovered from BCs and would have improved the initial management in 11/110 (10%) patients.