ObjectiveTo explore the clinical application of the comprehensive guidance technologies, such as cone beam computed tomography (CBCT), virtual bronchoscopic navigation (VBN), and superimposed high-frequency jet ventilator for respiratory control in the biopsy of peripheral pulmonary nodules (PPNs). MethodsThe clinical information of 3 patients with PPNs diagnosed by CBCT combined with VBN and superimposed high frequency superposition jet ventilator in Shanghai Changhai Hospital were retrospectively analyzed. Results Clinical data of 3 patients were collected. The average diameter of PPNs was (25.3±0.3) mm with various locations in left and right lung. The first nodule was located in the apex of the left upper lung, and the biopsy was benign without malignant cells. The lesion was not enlarged during the 5-year follow-up. The second one was located in the left lingual lung, and the postoperative pathology was confirmed as mucosa-associated lymphoma. The third one was located in the anterior segment of the right upper lung. After the failure of endobronchial procedure, percutaneous PPNs biopsy under CBCT combined with VBN was performed, and the pathological diagnosis was confirmed as primary lung adenocarcinoma. Postoperative pneumothorax complication occurred in the third patient with right lung compression rate approximately 20%. ConclusionsThe application of CBCT, combined with VBN and the superimposed high frequency jet ventilator for respiratory control can potentially improve the accuracy and safety in the diagnosis of PPNs. Multi-center clinical trials are needed to verify its further clinical application.