Purpose: The diagnosis of pulmonary fungal infections is challenging due to the difficulty of obtaining sufficient specimens. Endobronchialultrasound-guided transbronchial needle aspiration (EBUS-TBNA) needle rinse fluid has become an emerging diagnosticmaterial. This study evaluated the role of routine fungal culture from EBUS-TBNA needle rinse fluid, in addition to histopathologicexamination and fungal culture of EBUS-TBNA core tissue, in the diagnosis of pulmonary fungal infections. Materials and Methods: Among patients who underwent EBUS-TBNA, those with results for at least one of three tests (histopathologicexamination, fungal culture of EBUS-TBNA core tissue or needle rinse fluid) were included. Patients with a positive test weredivided into two groups (clinical fungal infection and suspected fungal contamination) according to their clinical assessment andtherapeutic response to antifungal. Results: Of 6072 patients, 41 (0.7%) had positive fungal tests and 9 (22%) were diagnosed as clinical fungal infection. Of the 5222patients who were evaluated using a fungal culture from EBUS-TBNA needle rinse fluid, 35 (0.7%) had positive results. However,only 4 out of 35 (11.4%) were classified as clinical fungal infection. Positive results were determined in 4 of the 68 (5.9%) evaluatedby a fungal culture of EBUS-TBNA core tissue, and all were diagnosed as clinical fungal infection. Conclusion: Routine fungal culture of EBUS-TBNA needle rinse fluid is not useful due to the low incidence of fungal infection andhigh rate of contamination. However, fungal culture of EBUS-TBNA core tissue and needle rinse fluid should be considered in patientswith clinically suspected fungal infection.