Background: Earlier reports on the detection of H. pylori DNA in gallbladder tissue of patients with cholelithiasis and cholecystitis gave discordant results. Our aim was to detect the presence of H. pylori DNA and to determine whether a correlation can be established with the biliary diseases. Methodology: The study included a total of 68 patients 20 to 79 years of age. Fifty-three of the participants were females, of whom 33 had cholelithiasis and 20 did not. Out of the 15 male patients, 8 were had cholelithiasis and 7 did not. Gallbladder tissue specimens were taken from all patients undergoing cholecystectomy and processed immediately for histology, culture and PCR. Results: Histological examination revealed that 36 (68%) of 53 females and 9 (60%) of 15 males had cholecystitis. PCR results detected H. pylori DNA in 15 (22%) of 68 samples but no bacteria were isolated in culture. The presence of few bacteria, the geographic distribution of H. pylori strains, and the bile milieu inhibitory effect might be some of the reasons for growth failure. Conclusions: In this study although H. pylori DNA was detected by PCR in gallbladder tissue of patients with cholecystitis, a clinical correlation with biliary disease could not be established because several conditions were difficult to meet as discussed in the text.