Background and Purpose: Optimal antibiotic prophylaxis is crucial to prevent postoperative infection in spinal surgery. Sufficient time above the minimal inhibitory concentration (fT > MIC) for relevant bacteria in target tissues is required for cefuroxime. We assessed cefuroxime concentrations and fT > MIC of 4 μg·ml⁻¹ for Staphylococcus aureus in the intrathecal (spinal cord and cerebrospinal fluid, CSF) and extrathecal (epidural space) compartments of the lumbar spine. Experimental Approach: Eight female pigs were anaesthetized and laminectomized at L3–L4. Microdialysis catheters were placed for sampling in the spinal cord, CSF, and epidural space. A single dose of 1500 mg cefuroxime was administered intravenously over 10 min. Microdialysates and plasma were obtained continuously during 8 h. Cefuroxime concentrations were determined by ultra-high-performance liquid chromatography. Key Results: Mean fT > MIC (4 μg·ml⁻¹) was 58 min in the spinal cord, 0 min in the CSF, 115 min in the epidural space, and 123 min in plasma. Tissue penetration was 32% in the spinal cord, 7% in the CSF, and 63% in the epidural space. Conclusion and Implications: fT > MIC (4 μg·ml⁻¹) and tissue penetration for cefuroxime were lower in the intrathecal compartments (spinal cord and CSF) than in the extrathecal compartment (epidural space) and plasma, suggesting a significant effect of the blood–brain barrier. In terms of fT > MIC, a single dose of 1500 mg cefuroxime seems inadequate to prevent intrathecal infections related to spinal surgery for bacteria presenting with a MIC target of 4 μg· ml⁻¹ or above.