Drinking water distribution system biofilms were investigated for the presence of hygienically relevant microorganisms. In this study we collected four PVC pipes from different Drinking water and studied the biofilm morphology, Characterizing the formation of biofilm on Polyvinyl chloride (PVC) pipe lines were analyzed by using the techniques, X-ray diffraction (XRD), Raman microscopy (RMS), X-ray fluorescence spectroscopy (XRF), Fourier transform infrared spectroscopy (FT-IR), and Scanning electron microscopy (SEM EDAX). Biofilm formation in drinking water PVC pipelines is due to many factors including pH, temperature, salt etc. In this study, microbiological aspects of biofilm formation and their role on electrochemical studies are presented. We report the design and evaluation of PCR primers 63f and 1387r for amplification of 16SrRNA genes from bacterial isolates, Phylogenetic analysis of partially sequenced 16SrRNA gene sequences revealed the diversity of bacterial and fungal strains in the PVC pipeline 7 different bacterial phyla were detected. In addition, localized biofilm of the PVC pipeline in the presence of the consortia was observed by scanning electron microscopy analysis and morphology was seen in EPI fluorescence microscopy, The dominant bacterial species identified in the PVC pipeline biofilm samples were Sphingobacterium sp respectively, several types of bacteria may be involved in biofilm arising and the potential role of each species in biofilm formation is discussed. Therefore, calcium carbonate and the allied calcium compounds are responsible for the biofilm formation in PVC pipes and these inferences may lead to develop effective water purification measures.