Earlier reports on the detection of Helicobacter DNA in the gallbladder tissue of patients with biliary diseases have shown discordant results. This study aimed to detect the presence of Helicobacter in gallstone, gallbladder tissue and bile specimens from subjects with H. pylori-positive gastritis with cholelithiasis. The presence of H. pylori in antrum biopsies was confirmed by rapid urease test and/or histopathological examination. DNA was extracted from gallbladder, bile and gallstone samples from 50 patients undergoing cholecystectomy. The presence of Helicobacter genus-specific DNA (16S rRNA genes) was determined by nested polymerase chain reaction assay. Helicobacter DNA was detected in the gallbladder tissue and bile of 28% and 18% respectively of the patients, but was not detected in any of the gallstones. These results do not rule out the possibility of Helicobacter infection as a contributing agent or cofactor in the development of biliary diseases.