Introduction Different approaches have been proposed for bacterial identification in patients with a suspected periprosthetic joint infection (PJI). If a one-stage procedure is considered, a higher rate of preoperative bacterial identification can be achieved if biopsy is included in the diagnostic work-up. The performance of open biopsy (OB) in the context of PJI has not been clearly determined yet. The purpose of this study was to determine the value of an OB added to two consecutive culture-negative joint aspirations during PJI workup. Materials and methods We retrospectively analyzed the OB data from a single institution. Patients under PJI work-up of the hip or knee with two culture-negative periprosthetic aspirations who underwent OB were included. Sensitivity and specificity were calculated using the musculoskeletal infection society (MSIS) criteria as gold standard. Patients undergoing urgent irrigation and debridement and patients with history of surgery to the affected joint in the prior 6 weeks were excluded. Results 126 patients were included in this study. 62 (49.2%) patients had prior revisions, 48 of them due to PJI. The sensitivity and specificity of OB was 69.4% and 89.1%, respectively. The OB procedure led to the identification of the causative germ in 50 out of 126 (40%) cases so they could undergo one-stage (septic) exchange. Conclusion The OB is a valuable resource if preoperative synovial fluid cultures are negative, a high suspicion of infection persists and a one-stage procedure is preferred. It intends bacteria identification and allows surgeons to evaluate prosthetic complications for further surgical procedures.