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Microbiological diagnosis is crucial for the appropriate management of implant-associated orthopedic infections (IAOIs). Sonication of biomaterials for microbiological diagnosis has not yet been introduced in routine clinical practice. Aim of this study was to describe the advantages and feasibility of this procedure in the clinical setting. We prospectively studied 56 consecutive patients undergoing revision because of IAOI and compared the sensitivity of sonication of explanted orthopedic implants with standard cultures. Patients were divided into two groups: those with foreign body infection (FBI, 15 patients) and those with prosthetic joint infection (PJI, 41 patients). Clinical, radiological and microbiological features were recorded. In the PJI group the sensitivity of sonication in detecting bacterial growth was higher than conventional culture (77% vs 34.1% respectively, p< 0.002), while no difference was observed in the FBI group (85.7% vs 86% respectively, p> 0.05). Coagulase-negative Staphylococci accounted for 90% of the bacteria detected by sonication. Moreover, we found that in the PJI group the sensitivity of sonication was not affected by the timing of antibiotic interruption before surgery. Sonication remains an important tool to improve microbiological diagnosis in PJIs, especially in patients who received previous antimicrobial treatment.