The most prevalent gastrointestinal gallstone disease and Gall Bladder Cancer is supposed to augment in the majority of ageing populations at risk in developing as well as developed countries. The exact mechanism of the cancer is not yet clearly understood. Total metagenome from the samples were extracted using the DNA Isolation kit according to the manufacturer’s instructions. DNA of culturable heterotrophic bacterial community was extracted from the cell biomass grown on R2A agar plate using DNeasy tissue kit as per manufacturer’s instructions. All DNA extractions were done in triplicate and pooled into one sample prior to use in PCR. PCR amplification of 16S rRNA genes was carried out. The amplified PCR products (~1500 bp) were gel purified, cloned into the pGEM-T® Easy vector and transformed into E. coli JM109 following the manufacturer’s instructions. Two clone libraries J254TP and J254PW were constructed from total metagenome and culturable metagenome respectively with clones having proper length of 16S rRNA gene fragment. PCR amplified product (5 µl) from each clone was digested separately with HaeIII and MspI in 20-µl reaction mixtures.All digests were analyzed by 2.5% agarose gel electrophoresis. ARDRA patterns were checked manually and each ribotype or Operational Taxonomic Unit (OTU) was defined as a group of clones that had similar enzyme restriction pattern. At least one representative clone from each dominant OTU was selected for sequencing of the 16S rRNA gene insert. Partial sequence of 16S rRNA gene fragment was determined on ABI 3730 machine. The retrieved 16S rRNA gene sequences from the GenBank were aligned along with the new sequences using ClustalW and phylogenetic analysis was done by MEGA 4.0. Experimental findings illustrate the presence of Staphylococcus, Micrococcus etc which might give a limelight and help us to explore the etiological factors for causing the disease.