Introduction: Diagnosis of bloodstream infections using bacteriological cultures suffers from low sensitivity and reporting delay. Advanced molecular techniques introduced in many laboratories provide rapid results and may show improvements in patient outcomes. This study aimed to evaluate the usefulness of a molecular technique, broad-range 16S rRNA PCR followed by sequencing for the diagnosis of bloodstream infections, compared to blood culture in different patient groups. Methodology: Conventional PCR was performed, using broad-range 16S rRNA primers, on blood cultures collected from different patients with suspected bloodstream infections; results were compared with those of blood culture. Results: Though blood culture is regarded as the gold standard, PCR evaluation showed sensitivity of 86.25%, specificity of 91.25%, positive predictive value of 76.67%, negative predictive value of 95.22%, and accuracy of 88.8%. Conclusions: Molecular assays seem not to be sufficient to replace microbial cultures in the diagnosis of bloodstream infections, but they can offer a rapid, good negative test to rule out infection due to their high negative predictive value.