Aim: The purpose of this in vitro study was to evaluate the effect of CO2 laser on the periodontally involved root surface, and to compare its efficacy with hydrogen peroxide, EDTA and citric acid on removal of smear layer. Methodology: 50 specimens from the proximal surface of periodontally involved extracted human teeth were used. Group A specimens received CO2 laser. Groups B, C and D were treated with 6% hydrogen peroxide, EDTA (pH 7.4), and citric acid (pH 1) respectively. The specimens were then scanned using Scanning Electron Microscopy. Results: The CO2 laser was not able to remove the smear layer on the sites that were irradiated for 0.2, 0.4 or 0.6 seconds at 3W power. Irradiation time of 0.8 seconds at 3W power was able to remove the smear layer, but the dentinal tubules were partially exposed. The surface irradiated for 1 second showed a flat appearance with many clear orifices of dentinal tubules. Irradiation time of 1.2 and 1.4 seconds produced surface charring and was totally ineffective in exposing the dentinal tubules. Hydrogen peroxide did not remove the smear layer completely. EDTA and citric acid were found to be effective in removing the smear layer and exposing the dentinal tubules, however the exposed dentinal tubules showed funnel shaped widening. Conclusion: Results suggest that CO2 laser produced increased exposure of dentinal tubules more effectively as compared to hydrogen peroxide, EDTA and Citric acid.