Background/Objectives: Gingival recession poses significant challenges in periodontal\r\ntherapy, particularly in procedures aimed at achieving predictable root coverage and long-\r\nterm stability of grafts. Conditioning of the root surface plays a crucial role in improving\r\nbiomaterial adhesion and facilitating periodontal regeneration. This in vitro study aimed\r\nto evaluate the morphological and microroughness alterations of root cementum following\r\ndifferent mechanical and chemical conditioning protocols commonly used in mucogingival\r\nsurgery. Methods: Forty extracted human single-rooted teeth were randomly allocated\r\ninto eight groups: untreated control, mechanical scaling alone, and scaling combined\r\nwith ethylenediaminetetraacetic acid (EDTA), citric acid, phosphoric acid, tetracycline,\r\ndoxycycline, or saline. Surface roughness was measured using contact profilometry, while\r\nstructural modifications were analyzed via scanning electron microscopy. Results: Sta-\r\ntistically significant intergroup differences (p < 0.05) were observed. Baneocin treatment\r\nproduced the most conservative changes, with limited surface roughness and minimal\r\nstructural alteration, whereas phosphoric acid, tetracycline, and EDTA caused pronounced\r\ndemineralization and surface porosity. Citric acid and doxycycline induced moderate\r\nalterations, with partial preservation of cementum integrity. The hypothesis assuming\r\nno surface or morphological changes was rejected. Conclusions: These findings indicate\r\nthat low-aggressiveness agents may achieve an optimal balance between surface decontam-\r\nination and cementum preservation, which is critical for enhancing graft integration and\r\nimproving clinical outcomes in root coverage surgery.