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The application of transbronchial lung cryobiopsy (TBLC) and uniportal and tubeless video-assisted thoracic surgery (UT-VATS) in the multidisciplinary diagnosis of interstitial lung disease (ILD) has not been demonstrated in real-world clinical practice. This prospective study included 137 patients with no definitive diagnosis who were the subject of two multidisciplinary discussion (MDD) sessions. As indicated in the first MDD, 67 patients underwent UT-VATS and 70 underwent TBLC. The specificity of biopsy information and its contribution to final MDD diagnosis were evaluated in the second MDD. The post-operative complications and hospitalization costs associated with the two biopsy methods were compared. UT-VATS was favored for patients initially diagnosed with idiopathic pulmonary fibrosis (IPF), bronchiolitis-associated interstitial lung disease (RB-ILD)/desquamative interstitial pneumonia (DIP) and undefined idiopathic interstitial pneumonia (UIIP), while TBLC was preferred for pulmonary lymphangioleiomyomatosis (PLAM) and pulmonary alveolar proteinosis (PAP). The spirometry parameters were better in patients who underwent UT-VATS than those who underwent TBLC. UT-VATS provided more specific pathological results than TBLC (85.7 vs 73.7%, p = 0.06). In patients initially diagnosed with UIIP, pathological information from UT-VATS was more clinically useful than that obtained from TBLC, although both tests contributed similarly to cases initially diagnosed as interstitial pneumonia with auto-immune features (IPAF)/connective tissue disease-related ILD (CTD-ILD). The safety of UT-VATS was comparable with TBLC although TBLC was cheaper during hospitalization (US$4,855.7 vs US$3,590.9, p < 0.001). multidisciplinary discussion decisions about biopsies were driven by current knowledge of sampling and diagnosis capacity as well as potential risks of different biopsy methods. The current MDD considered UT-VATS more informative than TBLC in cases initially diagnosed as UIIP although they were equally valuable in patients initially diagnosed with IPAF/CTD-ILD.