Endobronchial ultrasound-guided transbronchial mediastinal cryobiopsy (CryoEBUS) is an emerging minimally invasive technique that provides larger, better-preserved samples compared to conventional needle aspiration, improving suitability for histopathological and molecular testing. This retrospective study aimed to characterize the clinical profile, procedural aspects, and diagnostic yield of CryoEBUS in patients undergoing biopsy for mediastinal lesions. Fifty-nine patients underwent CryoEBUS. The mean age was 64.4 years (SD: 15.8), and 54% were men. Systemic arterial hypertension was the most common comorbidity (34%). The most frequent presenting symptoms were cough (66.1%) and chest pain (32.2%). CryoEBUS was performed for mediastinal lesion sampling, with an average of 1.5 lymph node stations biopsied per patient. Station selection was based on imaging characteristics (size ≥10 mm or fluorodeoxyglucose (FDG) uptake). Freeze time ranged from four to six seconds. Histopathology confirmed malignancy in 76% of cases, with adenocarcinoma being the most common subtype (42%). Diagnostic success, defined as the acquisition of a conclusive histopathological diagnosis, was achieved in 93% of cases. Minor complications occurred in 17%, primarily self-limited bleeding. CryoEBUS appears to be a safe and effective diagnostic tool for mediastinal lesion evaluation. Its ability to provide high-quality tissue samples supports its clinical utility, especially when molecular or immunohistochemical studies are required for diagnosis and treatment planning.