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Background and objective Mediastinal lymphoma or right upper lung cancer is a common cause of superior vena cava obstruction syndrome (SVCOS). Endobronchial ultrasound-guided needle aspiration (EBUS-TBNA) biopsy is applied for the diagnosis of mediastinal mass disease. The aim of this study is to explore the safety and feasibility of EBUS-TBNA biopsy under general anesthesia for the clinical diagnosis of SVCOS. Methods Twenty-five cases of SVCOS received EBUS-TBNA under general anesthesia between June 2012 and June 2013. The group consisted of 16 male and 9 female patients aged 33 years to 76 years, with a median age of 62.5. Results Twenty-four cases were confirmed to be of malignant pathology, and one case failed to yield a clear pathological diagnosis. No patient experienced any complications related to the operation, such as heavy bleeding and pneumothorax. Conclusion For patients presenting with SVCOS, EBUS-TBNA under general anesthesia is a safe and reliable inspection method of high diagnostic yield. This method can be used for routine examination when other means cannot obtain clear pathological diagnosis.