Endoscopic stenting is a palliative approach for the treatment of diseases involving biliary obstruction. Its major limitation is represented by stent occlusion, followed by life-threatening cholangitis, often requiring stent removal and replacement. Although it has been suggested that microbial colonization of biliary stents could play a role in the clogging process, the so far available data, particularly on the role of anaerobic bacteria, are not enough for a comprehensive description of this phenomenon. Our study was focused on the analysis of 28 explanted biliary stents by culturing, denaturing gradient gel electrophoresis and scanning electron microscopy to identify all the aerobic/anaerobic bacteria and fungi involved in the colonization of devices and to verify the ability of isolated anaerobic bacterial strains to form a biofilm in order to better understand the mechanisms of stent clogging.