Intramedullary nailing (IMN) has been frequently indicated to treat long bone open and closed fractures, but infection following internal fixation may have devastating consequences, with higher costs. Treatment of intramedullary nail-associated infections (IMNI) is challenging and based upon surgery and adequate antibiotic administration, which requires the correct identification of causative microorganisms. However, there have been difficulties for the microbial diagnosis of IMNI, as the peri-prosthetic tissue cultures may show no microbial growth, particularly in patients with previous use of antibiotics. Sonication have shown higher sensitivity and specificity for microbial identification on a variety of orthopedic implant-associated infections. Aim: To compare clinical and microbiological results and sensitivity for the pathogen identification obtained by conventional peri-implant tissue culture samples with culture of samples obtained by sonication of explanted IMN implants, among patients presenting IMNI of long bones. Methods: Longitudinal prospective cohort study performed at a tertiary public hospital, ongoing since August 2011. We analyzed all patients with indication for IMN implant removal, and orthopedic-implant associated infections was defined according to previous publications addressing osteosynthesis-associated infections (Yano 2014). Minimal of 2 samples from the peri-implant tissue were taken and sent under sterile conditions to the laboratory for culture. Statistical analysis was performed McNemar's test for related proportions. Results: We included 26 patients presenting clinical signs of IMNI, of which tissue and sonication cultures were performed for 26 (100%) and 20 (77%) patients, respectively. Among them, 88% were male, with mean age was 35.9 years (range, 19–59 yo). Causes of trauma were mainly motorcycle crashes accounting 54% of accidents; tibia and fibula were affected in 65% and 27%, respectively. Gustilo open fracture classification was grade II (35%) and IIIA (35%). First stage management with external fixation for fracture stabilization was performed in 75% of trauma patients. Sensitivity of peri-prosthetic tissue culture and sonication was 80.7% (21/26), and 95% (19/20) (p< 0.05), respectively. Only one infected patient presented negative tissue and fluid cultures. Gram-positive cocci were isolated in 75% and 79% in tissue and sonication fluid cultures, respectively. Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus sp., were isolated from tissue and sonication culture in 43.5% and 36.3%, 8.7% and 22.7%, 13% and 13.7%, respectively. Polymicrobial infection was diagnosed in 3.8% (1/26) and 15.8% (3/19), patients by tissue and sonication fluid cultures (p< 0,01), respectively. Conclusion: Sonication of retrieved infected intramedullary nails has the potential for improving the microbiological diagnosis of IMNI