Background: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a technique developed to allow mediastinal staging of lung cancer and also to evaluate intrathoracic lymphadenopathy. In a tuberculosis-endemic area, tuberculosis should be considered as an etiology of mediastinal lymphadenopathy. The aim of this study was to investigate the utility of the routine culture for tuberculosis from specimens of EBUS-TBNA. Methods: We prospectively performed routine culture for tuberculosis from aspiration or core biopsy specimens got from 86 patients who had undergone EBUS-TBNA due to mediastinal lymphadenopathy between March 2010 and March 2011. Results: A total of 135 lymph node aspiration and 118 core biopsy specimens were included in this analysis. We confirmed the malignancy in 62 (72.9%), tuberculosis in 7 (8.1%), sarcoidosis in 7 (8.1%), asperogillosis in 2 (2.3%) and pneumoconiosis in 2 (2.3%) patients. One lung cancer patient had pulmonary tuberculosis coincidentally and 5 patients had unknown lymphadenopathy. The number of positive culture for Mycobacterium tuberculsosis by EBUS-TBNA is 2 (1.5%) from 135 lymph node aspiration specimens and 2 (1.7%) from 118 core biopsy specimens. Out of eight patients confirmed with tuberculosis, only one patient had positive mycobacterial culture of aspiration specimen from EBUS-TBNA without histopathologic diagnosis. Conclusion: These results propose that routine culture for tuberculosis from EBUS-TBNA may not provide additional information for the diagnosis of coincident tuberculous lymphadenitis. However, if there is any possibility of tuberculous lymphadenopathy or pulmonary tuberculosis, it should be considered to perform EBUS-TBNA in patients who have negative sputum AFB smears or no sputum production.