Treatment guidelines for fracture nonunion differ based on the presence or absence of infection. Low-grade infections without preoperative clinical signs of infection are difficult to distinguish from aseptic cases. Membrane filtration of sonication fluid (MF) has been shown to be a useful method for identifying septic nonunion. Therefore, the aim of this study was to evaluate the diagnostic value of MF in differentiating low-grade infected nonunion from aseptic cases. A prospective multicenter clinical study enrolled 75 patients with femoral or tibial shaft nonunion with planned revision surgery and without clinical suspicion of infection. During revision surgery, tissue from the nonunion zone was sampled for culture and histopathology, and the implant for sonication with MF and colony forming unit (CFU) quantification. Infection was diagnosed according to the diagnostic criteria for fracture-related infection. The diagnostic performance of MF CFU count was evaluated by receiver operating characteristic (ROC) curve and compared with that of tissue culture (TC), sonication fluid broth culture (SFC), and Histopathological Osteomyelitis Evaluation Score (HOES). Fifty-three nonunion cases were aseptic, and 22 had a low-grade infection. ROC curve had an area under the curve of 0.84. The optimal CFU cutoff to discriminate between low-grade infected and aseptic nonunion was 11.1 CFU/10 mL sonication fluid with 64% sensitivity and 89% specificity. SFC showed a higher sensitivity of 82% but a lower specificity of 81%. The sensitivity and specificity of TC were 77% and 96%, respectively, and those of HOES were 9% and 87%, respectively. Implementation of MF in clinical diagnostics as an adjunct to TC may improve the differential diagnosis between low-grade infected nonunion and aseptic nonunion.