Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) was recently introduced for sampling of mediastinal masses and nodes, for staging of lung cancer, and for diagnosing other benign diseases including tuberculosis (TB). However, EBUS-TBNA has not been used to date to confirm the presence of multidrug-resistant TB (MDR-TB). We here describe the diagnosis of MDR-TB of a mediastinal lymph node by EBUS biopsy. MDR-TB had been transmitted to our patient from her son who had pulmonary TB, and the diagnosis of MDR-TB was confirmed by a restriction fragment length polymorphism analysis of the IS6110 segment of Mycobacterium tuberculosis isolates. Our findings highlight the use of EBUS-TBNA in diagnosing TB, including MDR-TB, and malignancies, in a TB-endemic area.