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Biofilm mode of growth is a strategy in microorganisms to survive harsh growth conditions. Although previous studies have established the ability of Enterococcus faecalis to survive postendodontic environmental conditions, the effect of such conditions on the ultrastructural and physiochemical features of E. faecalis biofilm has received less attention. This study aims to evaluate the effect of different growth conditions on the characteristics of E. faecalis biofilm on root canal, and the penetration of E. faecalis into dentinal tubules. Forty-five intact noncarious human maxillary molars were experimented under nutrient-rich, nutrient-deprived, aerobic, and anaerobic conditions for a period of 21 days. Scanning Electron Microscopy with Energy Dispersive X-ray microanalysis, Laser Confocal Scanning Microscopy and Light microscopic examinations were carried out. The microscopic analysis highlighted a distinct variation in the ultrastructure of the biofilms formed under different experimental conditions. The EDX microanalysis showed a significant increase in the levels of Calcium (Ca) in the biofilm structures formed under anaerobic nutrient-deprived condition (p < 0.001). The depth of bacterial penetration was significantly greater in nutrient-rich condition (p < 0.001). This study demonstrated distinct ultrastructural and physiochemical properties of the biofilms formed and dentinal tubular penetration of E. faecalis under different conditions.