In humans, free-hand computed tomography (CT)-guided biopsy is an accurate method to obtain a tissue sample. There are only a few reports of this technique in veterinary medicine. In the present study, 21 dogs and two cats underwent a free-hand CT-guided tissue-core biopsy (17 animals) or fine-needle aspiration (six animals) of a bone lesion. Two out of 17 tissue-core samples were also cultured. All 17 tissue-core biopsy samples were diagnostic (accuracy of 100%). Five out of six aspirates were diagnostic (accuracy of 83.3%). The overall accuracy was 95.7%. In one aspirate, cytologic quality was insufficient containing only blood. No major complications were encountered. Fourteen neoplastic, two infectious and six benign lesions were diagnosed. CT examination after intravenous contrast medium added useful information to avoid large vessels and to biopsy-viable tissue. Free-hand CT-guided tissue-core biopsy and aspiration appears to be a safe and very accurate procedure for use in the diagnosis of bone-associated diseases in small animals.