Enterococcus faecalis is the most common bacteria recovered from the root canals of teeth with persistent endodontic infection. They are facultative anaerobes, which are capable of surviving harsh environmental conditions, and are innately resistant to many antibiotics. Isolates from clinical samples are shown to be more resistant to antimicrobial agents. However, there are few investigations that studied the development of E. faecalis as biofilm. Objectives: the aim of the present study was to investigate the formation and environmental conditions that support E. faecalis growth as biofilm in root canal. Methods: Thirty-five human molar teeth were prepared by removing the crown and root tip to obtain a standard root length of 8 mm. The canal was enlarged to a size # 50 file and the smear layer was removed by treatment with 17% EDTA followed by 5.25% Sodium hypochlorite. Sterilized teeth samples were divided into two major groups. One group was kept in All Culture media (nutrient-rich condition), and the other in 1M PBS (nutrient-stress condition). The nutrient-rich group and the nutrient-stress group were further subdivided into aerobic and anaerobic subgroups, based on the type of incubation received. Scanning Electron Microscopy was used to observe the biofilm formation at different time intervals and under different experimental conditions. Results: The microscopic analysis revealed different stages in the biofilm formation by E. faecalis in root canal. The biofilm forming capability and the penetration of bacteria into dentinal tubules were found to vary under different experimental conditions. Conclusions: This study established stages of biofilm formation and supports the finding that the biofilm forming capability of E. faecalis in root canal is influenced by nutritional and environmental conditions. This work was supported by NUS Academic Research Grant FY2003-FY2004.