Microbiological and histological analysis of a bone specimen is the criterion standard for diagnosing osteomyelitis. The role of percutaneous bone biopsy (PBBx) in diabetic patients with possible osteomyelitis remains controversial: does PBBx improve diagnostic accuracy, provide important microbiological information, and can it be performed safely? We reviewed a series of 34 diabetic patients at one institution with suspected osteomyelitis who underwent (a) clinical evaluation, (b) wound culture, (c) imaging with standard X-rays, 99Tc-bone scans, and 111In-labelled white blood cell scans, and (d) fluoroscopically-guided PBBx. Osteomyelitis was diagnosed in 15 of 34 patients (44%), defined as PBBx or surgical bone specimens positive by culture or histology, or imaging and prolonged clinical follow-up. The overall sensitivity of PBBx for the diagnosis of osteomyelitis was 73% (11/15 patients diagnosed). Sensitivity increased to 89% among the 9 patients with osteomyelitis who had received ≤20 days of antibiotic therapy prior to biopsy. Four of eleven patients (36%) with intermediate probability imaging studies were subsequently diagnosed with osteomyelitis. PBBx identified 3 of these 4 patients with osteomyelitis. The only false-negative occurred in a patient who had received 30 days of antibiotics prior to biopsy. Among patients with osteomyelitis and positive bone cultures, the causative organism identified by PBBx was not cultured from the overlying wound in 4 of 11 instances (36%). S. aureus was cultured from 55% of bone biopsies. There were no immediate or long-term complications of PBBx. Percutaneous bone biopsy is safe, highly specific and moderately sensitive for diagnosing osteomyelitis in patients with diabetic foot ulcers, particularly in the absence of prolonged antibiotic therapy prior to biopsy. PBBx may be most rationally utilized when the results of imaging studies are inconclusive (intermediate pre-test probability) or when an uncommon etiologic organism for osteomyelitis is cultured from the overlying wound (in order to direct antibiotic therapy).