Amplification of the universal 16S rRNA gene using PCR has improved the diagnostic yield of microbiological samples. However, no data have been reported on the reliability of this technique with venous access ports (VAPs). We assessed the utility of 16S rRNA PCR for the prediction of VAP-related bloodstream infection (VAP-RBSI). During a 2-year period, we prospectively received all VAPs removed by interventional radiologists. PCR and conventional cultures were performed using samples from the different VAP sites. We compared the results of PCR with those of conventional culture for patients with confirmed VAP-RBSI. We collected 219 VAPs from 219 patients. Conventional VAP culture revealed 15 episodes of VAP-RBSI. PCR revealed a further 4 episodes in patients undergoing antibiotic therapy which would have gone undetected using conventional culture. Moreover, it had a negative predictive value of 97.8% for the prediction of VAP-RBSI when it was performed using biofilm from the internal surface of the port. In conclusion, universal 16S rRNA PCR performed with samples from the inside of VAPs proved to be a useful tool for the diagnosis of VAP-RBSI. It increased detection of VAP-RBSI episodes by 21.1% in patients undergoing antibiotic therapy whose episodes would have gone undetected using conventional culture. Therefore, we propose a new application of 16S rRNA PCR as a useful tool for the diagnosis of VAP-RBSI in patients receiving antibiotic therapy.