The use of cardiopulmonary bypass (CPB) during cardiac surgery causes regional ventilation-perfusion mismatch, contributing to regional disturbances in antibiotic penetration into lung tissue. Ventilation-perfusion mismatch is associated with postoperative pneumonia, a frequent and devastating complication after cardiac surgery. In this prospective clinical animal study, we performed in vivo microdialysis to determine the effect of CPB on regional penetration of levofloxacin (LVX) into lung tissue. Six pigs underwent surgery with CPB (CPB group), and another six pigs underwent surgery without CPB (off-pump coronary artery bypass grafting; OPCAB group). LVX (750 mg) was administered intravenously to all pigs immediately after surgery. For regional measurements of LVX in pulmonary concentrations, microdialysis probes were inserted in both lungs of each pig. Pigs were placed in the right lateral position. Time versus concentration profiles of unbound LVX were measured in the upper and lower lung tissue and plasma in all pigs. In all pigs, maximum concentrations (Cmax) of LVX were significantly lower in the upper lung than in the lower lung (OPCAB, P = 0.035; CPB, P < 0.001). Median Cmax of LVX showed a significant difference in the upper versus lower lung in the CPB group (P < 0.05). No significant difference was found in the median Cmax of LVX in the upper and the lower lung in the OPCAB group (P = 0.32). Our data indicate that CPB affects perioperative regional antibiotic penetration into lung tissue. Common clinical antibiotic dosing schemes should be reevaluated in patients undergoing coronary artery bypass grafting with CPB.