Large-scale nosocomial outbreaks of Serratia marcescens septicaemia in Japan have had a fatality rate of 20–60% within 48 h. As a countermeasure, a real-time PCR assay was constructed for the rapid diagnosis of S. marcescens septicaemia. This assay indeed detected S. marcescens in clinical blood specimens (at ca. 102 CFU ml−1), at a frequency of 0.5% in suspected cases of septicaemia. In mice, the assay provided estimates of blood S. marcescens levels at various infectious stages: namely, 107 to 108 CFU ml−1 at a fatal stage (resulting in 100% death), 104–105 CFU ml−1 at a moderately fatal stage (resulting in 50% or more death), and <103 CFU ml−1 at a mild stage (resulting in 100% survival), consistent with actual CFU measurements. Blood bacterial levels could be an important clinical marker that reflects the severity of septicaemia. The simultaneous detection of S. marcescens and the carbapenem resistance gene was also demonstrated.