The purpose of this study was to explore the potential of confocal laser scanning microscopy (CLSM) for in situ identification of live and dead Enterococcus faecalis in infected dentin. Eight cylindrical dentin specimens were infected with Enterococcus faecalis in BHI for 21 days. After the experimental period, the specimens were stained with fluorescein diacetate (FDA) and propidium iodide (PI) or acridine orange (0.01%) and analyzed by CLSM. Two noninfected dentin specimens were used as negative controls. CLSM analysis shows that the discrimination between viable (green) and dead (red) bacteria in infected dentinal tubules could be observed after staining with FDA/PI. Acridine orange was able to show metabolic activity of the E. faecalis cells inside the dentinal tubules showed by its red fluorescence. The viability of bacteria in infected dentin can be determined in situ by CLSM. FDA/PI and acridine orange are useful for this technique.