BACKGROUND: Chemical root surface conditioning has been introduced to detoxify, decontaminate, and demineralise the root surfaces affected by periodontitis, thereby removing the smear layer and exposing the collagenous matrix of dentin and cementum. The aim of the study is to compare the effect of 0.2 hyaluronic acid gel and 24 EDTA gel as root conditioning agents on instrumented root surfaces of periodontally involved teeth using a scanning electron microscope (SEM). MATERIALS AND METHODS: In the present study, periodontally affected single-rooted teeth were selected. With a Gracey curette (5/6), root planing of root surfaces was done. A total of 30 samples were obtained from the cervical two-thirds of the root using hard tissue microtome. The samples were divided into three groups: GROUP - A: only scaling and root planing (SRP), GROUP - B: SRP + 24 EDTA, GROUP - C: SRP + 0.2 hyaluronic acid. The respective conditioning agents were applied on the specimens by the passive burnishing technique with a cotton pellet for 3 minutes. The samples were examined using an SEM. The results obtained were recorded and statistically analysed. RESULTS: The results revealed that the root conditioning agents used in this study were effective in removing the smear layer and increasing the total number and diameter of the dentinal tubules. Among them, 24 EDTA was more effective than 0.2 hyaluronic acid ( P value < 0.05 - statistically significant). CONCLUSION: The use of root conditioning agents increased the number of dentinal tubules opening. The efficacy of 24 EDTA seems to be higher than that of 0.2 hyaluronic acid. This record is sourced from MEDLINE/PubMed, a database of the U.S. National Library of Medicine