While the benefits of sonication for improving periprosthetic joint infection (PJI) are well-documented, its potential therapeutic effect against bacterial biofilm remains unstudied. This study aimed to investigate the safety and efficacy of a novel nanoparticle ultrasonication process on methicillin-resistant Staphylococcus aureus (MRSA) bacterial biofilm formation in a PJI rat model. This novel ultrasonication process was designed to remove attached bacterial biofilm from implant and peri-articular tissues, without damaging native tissues or compromising implant integrity. Twenty-five adult Sprague-Dawley rats underwent a surgical procedure and were colonized with intra-articular MRSA, followed by the insertion of a titanium screw. Three weeks after the index surgery, the animals received a second procedure during which the screws were explanted, and soft tissue was sampled. The intraoperative use of the nanoparticle sonication treatment was employed to assess the device's safety, while ex vivo treatment on the retrieved tissue and implants was used to evaluate its efficacy. Clinical and histological assessments did not indicate any macro- or micro-damage to the host tissue. Sonication of the retrieved tissues demonstrated an average bacterial removal of 2 × 103CFU/mL and 1 × 104CFU/gram of tissue. Compared to the standard-of-care group (n = 10), implants treated with sonication (n = 15) had significantly lower remaining bacteria, as indicated by crystal violet absorbance measurements (P = 0.012). This study suggests that nanoparticle sonication technology can successfully remove attached bacterial biofilms from explanted orthopedic hardware and the joint capsule, without negatively affecting native tissue. The study provides initial results supporting the potential of nanoparticle sonication as an adjuvant treatment option during a DAIR (debridement, antibiotics, and implant retention) procedure for PJI, paving the way for future clinical trials.