ABSTRACT Background Whether sonographic features of mediastinal lymph nodes can differentiate malignancy from tuberculosis remains unclear. Research design and methods We retrospectively identified subjects with a confirmed diagnosis of tuberculosis or malignancy on endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). Our primary objective was to compare the endosonographic characteristics of lymph nodes on EBUS between tuberculosis and malignancy. Our secondary objective was to assess the diagnostic performance of endosonographic characteristics in predicting malignancy. Results We included 774 subjects (1,498 lymph nodes) with a confirmed diagnosis of tuberculosis (n = 497) or malignancy (n = 277). Distinct lymph node margins (84.1% vs. 93.8%, P < 0.001) and coagulation necrosis sign (11.2% vs. 29.8%, P < 0.001) were less common in malignancy than tuberculosis. The absence of central hilar structure had the highest sensitivity (92.1%) for malignancy. Endosonographic characteristics had poor specificity for malignancy(round shape and coagulation necrosis sign, 77.3% and 70.2%. In multivariate analysis, coagulation necrosis sign was associated with a lower odds of malignancy (odds ratio 0.45 [95% confidence intervals, 0.21–0.95]). Conclusions Endosonographic characteristics, such as round shape and the coagulation necrosis sign, are not specific for malignancy in high tuberculosis prevalence areas.