Rapid detection of bloodstream infections is an important issue for a better patient outcome. The aim of our study was thus to evaluate the LightCycler SeptiFast assay for diagnosis of bloodstream pathogens in a tertiary hospital in Western Austria. The 71 blood samples of 61 patients with presumed sepsis were investigated and compared with conventional blood culture system results. In both assays, 51 samples (71.8 %) were negative. In 20 positive samples (28.2 %), 10 different pathogens were detected by either blood culture system or SeptiFast assay or by both methods. Five samples were positive in both assays. The agreement rate of blood culture system and SeptiFast assay was 78.9 %, the negative predictive value of SeptiFast assay versus blood culture system was 0.94, sensitivity was 0.63, and specificity 0.81. In 12 samples where a positive SeptiFast assay and a negative blood culture system result were obtained, the same pathogens as identified by SeptiFast assay were detected in samples from other body sites suggesting a correct positive detection. In 11.3 % of cases, the SeptiFast assay resulted in an adjustment of the patients' therapy. In 3 samples, the blood culture assay was positive whereas the SeptiFast assay yielded negative results. In two of these cases, the pathogens involved were not included in the SeptiFast detection list, in the third case SeptiFast assay failed to detect Candida glabrata.Thus we recommend the SeptiFast assay as a valuable tool for rapid diagnosis of bloodstream infections in addition to, but not as replacement for, the blood culture test.