We report a 45-year-old man with HIV/AIDS who developed mediastinal lymphadenopathy caused by Nocardia asteroides infection that was diagnosed by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). He was an untreated HIV-infected man who was admitted to our hospital because of Pneumocystis pneumonia and Cytomegalovirus pneumonia. After treatment for pneumonia, cough and fever recurred and chest computed tomography revealed subcarinal lymphadenopathy with rim enhancement. To identify the etiology, we performed EBUS-TBNA and obtained purulent exudates which contained N. asteroides. EBUS-TBNA is a useful and safe technique for the diagnosis of mediastinal infectious lymphadenopathy of unknown origin.