Abstract Bloodstream infections are associated with high morbidity and mortality. Blood culture is considered to be the most important tool in their diagnosis. In this study we compared standard blood culture with two different molecular techniques (Prove-it™ Sepsis and LightCycler® SeptiFast M grade) to detect bacterial species and fungi in the blood of septic patients. Both methods showed significant agreement with the results obtained by blood culture. The results of these studies suggest that both the LightCycler SeptiFast and Prove-it Sepsis tests can be used for the rapid and specific diagnosis of bacteraemia and bloodstream infections. It is important, however, to emphasize that molecular methods cannot replace blood cultures, as the latter allow the evaluation of the antimicrobial susceptibility of isolated microbes.