The superior performance of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in the diagnosis and staging of malignancy has been demonstrated, with some investigators suggesting the same for sarcoidosis. The role of EBUS-TBNA in the diagnosis of histoplasmosis is not clear, however. In this study we estimate the diagnostic yield of conventional TBNA (cTBNA) and EBUS-TBNA for the diagnosis of histoplasmosis in patients with mediastinal lymphadenopathy. Retrospective chart review was conducted on 452 consecutive patients who underwent cTBNA or EBUS-TBNA for mediastinal lymphadenopathy from January 1, 2005 to December 31, 2014 at Methodist Le Bonheur Healthcare-affiliated hospitals in Memphis, Tennessee. Data collection included demographic information, reason for the procedure, size of the lymph nodes, procedures performed, and the final diagnosis. Among 452 cases reviewed, 146 underwent cTBNA and 306 underwent EBUS-TBNA. Final diagnoses include malignancy (41.5%), sarcoidosis (11.2%), and histoplasmosis (8.1%). Among 146 patients who underwent cTBNA, a final diagnosis was obtained by this modality in 58 patients (39.7%). The diagnostic rate for cTBNA for malignancy was 68% (40/59), 30% (4/13) for sarcoidosis, and 43% (6/14) for histoplasmosis. In 306 patients who underwent EBUS-TBNA, 188 had a final diagnosis (61.4%) obtained by this modality. For EBUS-TBNA, the diagnostic rates were 79.5% (101/127) for malignancy, 74% (28/38) for sarcoidosis, and 78% (18/23) for histoplasmosis. EBUS-TBNA had a higher yield than cTBNA for the diagnosis of histoplasmosis. Clinicians practicing in areas with a high prevalence of histoplasmosis and sarcoidosis should use EBUS-TBNA, whenever available, for this reason.