The aim of this study was to evaluate the effect of photoactivated chemotherapy (PACT) on Enterococcus faecalis (E. faecalis) biofilms in root canals using an 90% isopropanol (IPA)-based photosensitizer and removing excess photosensitizer before light incubation. Three hundred and seven extracted human teeth with one root canal were infected with E. faecalis for 72 h and treated in groups: IPA irrigation; PACT; PACT and final rinse with IPA; PACT with photosensitizer removal using either 0.9% NaCl solution or sterile paper points or both; PACT using IPA-based photosensitizer with and without a final rinse of IPA. Root canals were sampled using sterile paper points and dentin chips collected from the root canal walls. Additionally, SEM (Scanning Electron Microscopy) images of the specimens were taken to evaluate the root canal walls for residue bacterial contamination. In all antimicrobial treatment groups treatments E. faecalis counts were significantly reduced in the root canals. Using IPA-based photosensitizer the antimicrobial effect of PACT was significantly enhanced. Irrigation with IPA alone or after PACT significantly increased the antimicrobial effect compared to PACT alone. The collected dentin chips revealed the highest amount of culture negative root canals (10%) after PACT using IPA-based photosensitizer. In the other groups, the culture negative samples ranged from only 0-2 specimens of 30 specimens. REM images show remaining E. faecalis cells on the root canal wall and inside dentin tubules. Using IPA-based photosensitizer significantly enhanced the antimicrobial effect of PACT against E. faecalis in the root canals.