The feasibility of the transtracheal approach to the thoracic cavity has been demonstrated, but surgical lung biopsy via the tracheal approach still remains a challenge. This study aimed to evaluate the feasibility and outcome of transoral surgical lung biopsy under a single preoperative dose of parenteral antibiotics. Transoral thoracoscopy and surgical lung biopsy were performed for 10 anesthetized dogs after a single intravenous injection of cefazoline (20 mg/kg). A 12-mm transoral incision was created on the vestibulum, and a homemade metallic tube was advanced into the thoracic cavity via the pretracheal and substernal space under endoscopic guidance. After thoracic exploration, surgical lung biopsy was performed using an electrosurgical snare with a flexible bronchoscope inserted through the metallic tube. The resection margin of the lung was secured with a homemade endoloop. The animals were killed by day 14 after the surgery for gross and histologic evaluations. The thoracic cavity was evaluated and lung biopsy was performed successfully (for 3 lobes in the right lung and 2 lobes in the left lung) in 9 of the 10 dogs. Neither mortality nor intraoperative complications were observed. The average time for the transoral thoracoscopy and surgical lung biopsy was 133.5 min. Postmortem examination showed complete healing, with fibrosis and moderate adhesion over the resection margin. No evidence of either mediastinitis or intrathoracic infection was observed. This study showed the feasibility of transoral thoracoscopy and surgical lung biospy in dogs. Moreover, single-dose prophylaxis with cefazoline in transoral surgical lung biopsy was found to be effective in preventing potential infection.