Aim of the study was to investigate the value of serum and bile neutrophil gelatinase-associated lipocalin (NGAL) for distinguishing malignant strictures caused by cholangiocarcinoma (CCA) or pancreatic cancer from benign biliary strictures. The study was performed prospectively on patients admitted for endoscopic or radiologic biliary decompression. Forty patients with dilated biliary ducts, including 16 cases of CCA, 6 cases of pancreatic cancer, and 18 cases of benign biliary stricture were enrolled. Their sera and bile were collected to measure NGAL. Routine biochemistry including measurement of serum levels of carbohydrate antigens (CA) 19-9 and carcinoembryonic antigen (CEA) was also performed. The serum CA19-9, serum CEA, and bile NGAL levels were significantly increased in patients with malignant strictures as compared with patients with benign biliary diseases. Serum NGAL had no significant value for discriminating between malignant and benign biliary strictures. Bile NGAL levels had a receiver characteristic area under the curve of 0.74, sensitivity 77.3, and specificity 72.2% for discriminating between pancreatobiliary cancer and benign biliary diseases. Bile NGAL and serum CA19-9 were independent parameters and their combined use improved diagnostic accuracy (sensitivity 91%, negative predictive value 85.7%). We conclude that measurement of biliary, but not serum NGAL, may differentiate malignant pancreatobiliary from benign biliary strictures, serving as a complementary biomarker for serum CA19-9.