This study analyzed the performance of different molecular technologies along with blood culture (BC) in the diagnosis of bloodstream infections (BSI) in patients from internal medicine wards - including intensive care units (ICUs) - and the emergency room. Patients with systemic inflammatory response syndrome were prospectively included. BCs and EDTA whole blood were obtained simultaneously. The latter was analyzed by PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS; IRIDICA BAC BSI assay, Abbott) and by SeptiFast (Roche). Cases were classified as BSI according to adapted European Centre for Disease Prevention and Control criteria. Out of 462 analyzed episodes, 193 with valid test results fulfilled the inclusion criteria and were further evaluated. Sixty-nine (35.8%) were classified as BSI. PCR/ESI-MS showed a significantly better overall performance than BC (p = 0.004) or SeptiFast (p = 0.034). Only in patients from the ICU the performance of SeptiFast was comparable to that of PCR/ESI-MS. Mainly due to the negative effect of antimicrobial pre-treatment on BC results, the cumulative performance of each of the molecular tests with BC was significantly higher than that of BC alone (p < 0.001). SeptiFast and in particular the broad-range pathogen detection system PCR/ESI-MS proved to be an essential addition to BC-based diagnostics in BSI.