Computed tomography (CT)-guided biopsy of the spine is considered a safe, accurate, and relatively inexpensive examination technique. Our purpose was to determine the diagnostic accuracy of CT-guided biopsies exclusively for vertebral osteomyelitis. A retrospective study was performed from a consecutive series of 72 patients with confirmed vertebral osteomyelitis with 46 CT-guided biopsies performed in 40 patients. Biopsy specimens were sent for bacteriologic and cytologic analysis. An adequate specimen for microbiologic examination was not obtained in one case and not enough sample for additional pathologic examination in 17 cases. The mean age of patients was 58 years, with a range of 1-88 years, including 24 men and 16 women. The level of spinal biopsy was thoracic in 18 (40%) and lumbar in 28 (60%). The analysis revealed the infection agent in 20 cases (43% sensitivity). Diagnostic rates obtained in patients with previous antibiotic treatment were significantly lower (23% vs. 60%, p = 0.013). Computed tomography-guided fine-needle aspiration biopsy is an important tool in the diagnostic evaluation of vertebral osteomyelitis. However, this technique yields a lower diagnostic rate than previously reported biopsy of neoplastic vertebral lesions, especially if performed in patients with previous antibiotic treatment.