Real-time PCR methods are able to rapidly detect a wide panel of microorganisms. These methods are of interest in critically ill patients to determine the presence of bacteria in the blood and other biological samples, especially in those patients with prior antimicrobial treatment. In intensive care unit (ICU), the LightCycler SeptiFast (LC-SF) Test provides 1.5 to 2 fold higher positivity rate compared with conventional blood cultures. Although identification of the bacterium by LC-SF is rapid and sensitive, susceptibility test could not be performed using this technique, except the methicillin- resistance for Staphylococci. The conventional cultures remain necessary for samples in ICU because of the high incidence of multidrug-resistant bacteria and the need for antimicrobial susceptibility of the bacterium to treat the patient correctly. A negative result for a Gram positive or negative bacterium allows deescalating the initial antimicrobial treatment, and decreasing the pressure of selection. Moreover, it is necessary to understand and interpret a DNA signal knowing that a dead bacterial material may be detected in a patient without any infection. What is the clinical relevance of bacterial DNA present in the blood and does the DNAemia found reflect true infection? Cost-effectiveness of the real-time PCR should be determined. Meanwhile, this test should be restricted to severe clinical situations, especially ICU patients with severe sepsis. In the future, real-time PCR tests should include more pathogens and antimicrobial resistant targets.