To assess diagnostic performance of CT-guided percutaneous needle bone biopsy (CTNBB) in patients with suspected osteomyelitis and analyze whether certain clinical or technical factors were associated with positive microbiology results. All CTNBBs performed in a single center for suspected osteomyelitis of the appendicular and axial skeleton during 2003-2018 were retrospectively reviewed. Specific inclusion criteria were clinical and radiologic suspicion of osteomyelitis. Standard of reference was defined using outcome of surgical histopathology and microbiology culture and clinical and imaging follow-up. Technical and clinical data (needle size, comorbidities, clinical factors, laboratory values, blood cultures) were collected. Logistic regression was performed to assess associations between technical and clinical data and microbiology biopsy outcome. A total of 142 CTNBBs were included (46.5% female patients; age ± SD 46.10 y ± 22.8), 72 (50.7%) from the appendicular skeleton and 70 (49.3%) from the axial skeleton. CTNBB showed a sensitivity of 42.5% (95% confidence interval [CI], 32.0%-53.6%) in isolating the causative pathogen. A higher rate of positive microbiology results was found in patients with intravenous drug use (oddsratio [OR]= 5.15; 95% CI, 1.2-21.0; P= .022) and elevated white blood cell count ≥ 10× 109/L (OR= 3.9; 95% CI, 1.62-9.53; P= .002). Fever (≥ 38°C) was another clinical factor associated with positive microbiology results (OR= 3.6; 95% CI, 1.3-9.6; P=.011). CTNBB had a low sensitivity of 42.5% for isolating the causative pathogen. Rate of positive microbiology samples was significantly higher in patients with IV drug use, elevated white blood cell count, and fever.