Purpose: Diabetic foot ulcer with osteomyelitis is notorious with its complexity and healing difficulties. Bone biopsy is considered to be the gold standard method of guidance for antibiotic therapy. However, it is often replaced by cultures of ulcer swabs or by superficial samples because of the technical difficulties and possible adverse events. In this study, we compared microbiologic results of bone biopsy with those of superficial tissue biopsy or swab culture to investigate concordance and diagnostic value in bone involved diabetic foot ulcers. Methods: This study involved 106 patients with diabetic foot ulcers who showed positive results in bone probing test. Tissue samples for microbiologic tests were collected from all the patients by using superficial cotton swab, superficial tissue biopsy, and bone biopsy. The microbiologic results of bone biopsy were compared with swab culture and superficial tissue biopsy statistically. Results: The positive predictive value of bone probing test for underlying osteomyelitis was 82.1%. Microbiology of the bone biopsy showed same results with those of the swab culture and superficial tissue in 64% and 63%, respectively. Statistical analysis demonstrated that the microbiology of the swab culture or superficial tissue did not coincide with that of the bone biopsy. Conclusion: These results suggest that the microbiologic results of superficial tissue or swab culture do not coincide with those of bony tissue. To select appropriate antibiotic regimen for diabetic ulcer with bone involvement, the specimen for the microbiologic test should be obtained from involved bone.