Background and objective:We evaluated the usefulness of acid-fast bacilli (AFB) culture and Mycobacterium tuberculosis (MTB) polymerase chain reaction (PCR) of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) needle rinse fluid for diagnosing tuberculous lymphadenitis. Methods: EBUS-TBNA needle rinse fluid was routinely used for AFB culture and MTB PCR. The patients were categorized according to the pre-procedural diagnosis (Group A, suspected/histology-confirmed lung cancer; Group B, extrapulmonary malignancy; and Group C, other benign diseases). Results: Of the 4672 subjects, 104 (2.2%) were diagnosed with tuberculous lymphadenitis; 1.0%, 4.6% and 12.7% of Group A, B and C, respectively. Tuberculous lymphadenitis was diagnosed in 0.2%, 1.0% and 4.5% Group A, B and C patients, respectively, by histopathology. On addition of AFB culture to histopathology, tuberculous lymphadenitis was diagnosed in 1.0%, 4.4% and 10.3% of Group A, B and C patients, respectively (P < 0.001, P = 0.001 and P = 0.005, respectively). On addition of MTB PCR to histopathology, tuberculous lymphadenitis was diagnosed in 0.4%, 1.9% and 8.8%, respectively (Group C; P = 0.029). Conclusion: Routine AFB culture of needle rinse fluid was useful to increase the diagnostic yield of tuberculous lymphadenitis for all subjects who underwent EBUS-TBNA regardless of pre-procedural diagnosis in an intermediate tuberculosis (TB)-burden country. However, MTB PCR was only useful in subjects with pre-procedural diagnosis of benign pulmonary diseases.The routine acid-fast bacilli (AFB) culture of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) needle rinse fluid is useful to increase the diagnostic yield of tuberculous lymphadenitis in an intermediate tuberculosis (TB)burden country.