BACKGROUND Differentiation of benign and malignant biliary strictures plays a pivotal role in managing biliary strictures. Brush cytology via endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) are two diagnostic methods. In the present study, we aimed to compare the accuracy of the results of EUS-FNA and ERCP-based sampling of biliary strictures. METHODS In a prospective study, between January 2019 and March 2020, patients with indeterminate biliary strictures who had no history of hepatobiliary surgery, opium usage, cancer of pancratobiliary system, and acute liver disease were selected. They underwent EUS and ERCP in the same session. They were followed up for 6 months, and the sensitivity, specificity, positive and negative predictive values, and accuracy of these imaging modalities were compared. RESULTS A total of 60 patients were enrolled. 28 lesions were located in the distal and 32 lesions in the proximal parts of the biliary tree. 55 malignant and 5 benign lesions were diagnosed. The sensitivity and accuracy of EUS-FNA and ERCP tissue sampling were 78.2% and 80.0% versus 50.9% and 55.0%, respectively ( = 0.024). The combination of both methods improved the sensitivity and accuracy to 85.5% and 86.7%, respectively. Regarding the location, EUS-FNA is superior to ERCP-brush cytology in diagnosing proximal lesions with sensitivity and specificity of 73.3% and 75.0% vs. 50.0% and 53.1%, respectively ( = 0.04). CONCLUSION EUS-FNA is superior to ERCP brushing in the diagnosis of indeterminate biliary strictures, particularly in distal lesions. Combining ERCP brushing and EUS-FNA improves the diagnosis accuracy.