Background: Gallbladder stores and concentrates bile.Various factors influence the formation of gallstones.Bile infection is one of the important factors amongst all the other factors. There is a specific correlation between spectra of organisms in bile and biochemical composition of gallbladder calculi. Methods: Present study was conducted to assess the same where we studied the microbiology of bile and analysed the composition of gallstones by specific set techniques.Result:Infectious factor plays an important role in formation of pigment gallstones as compared to cholesterol and mixed stones most common bacteria being Escherichia coli. Conclusion:Moynihan’s aphorism that “gallstone is a tombstone erected in the memory of the organism within it” is true even today and can be concluded from our study too, Rate of bile infection increases with increasing age. Hence collecting bile samples for culture study and determining biochemical composition of gallbladder calculi should be emphasised in our routine surgical practice.