Microorganism survived in the root canal after root canal cleaning and shaping procedure is a main cause of root canal treatment failure. There are several mechanisms for the bacteria to survive in the root canal after chemomechanical preparation and root canal irrigation. Bacteria organized as biofilm has been suggested as an etiology of persistent periapical lesion. Recent studies were focus on removal of Enterococcus faecalis biofilm due to the report that the persistence of this bacteria after root canal treatment may be associated with its ability to form biofilm. Several investigations demonstrated that current root canal treatment protocol including use of NaOCl, EDTA and Chlorhexidine as irrigants is quite effective in eliminating E. faecalis biofilm. However, this microorganism still can survive in inaccessible areas of root canal system and evade host immune response, suppress immune activity and produce biofilm. Up to date, there is no possible clinical method to completely get rid of bacteria from the root canal. Once the root canal treatment failure occurred, and conventional treatment incorporating current therapeutic protocol has failed, periapical surgery or extraction should be considered rather than prolong the ineffected retreatment procedure.