Aim: The present study aimed to evaluate the effect of various root-conditioning agents on root surfaces that are periodontally affected. Materials and methods: A total of 90 human teeth having single root that were extracted because of chronic periodontitis were chosen. The extracted teeth were cleaned of saliva and blood using a soft-bristled brush and distilled water. The investigational groups were categorized into group I—doxycycline HCl, group II—ethylenediaminetetraacetic acid, and group III—tetracycline HCl. Samples were readied for histological study by scanning electron microscope (SEM). The SEM was used to assess the effectiveness of smear layer removal, amount of patent dentinal tubules out of the totality of dentinal tubules present, and the appreciation of collagen fiber-like structures inside the intertubular area. Results: The highest efficacy for smear layer removal was seen for group III—tetracycline HCl samples (1.80 ± 0.148) followed next by group II— EDTA (1.36 ± 0.230), and group I—doxycycline HCl (1.30 ± 0.283). The highest number of patent dentinal tubules were seen in group III—tetracycline HCl (44.50 ± 0.18) followed immediately by group II—EDTA (38.10 ± 0.42), and group I—doxycycline HCl (34.90 ± 0.23). The highest number of appreciation of collagen-like structures was recorded in group III—tetracycline HCl (2.64 ± 0.04) followed next by group I—doxycycline HCl (1.88 ± 0.10) and group II—EDTA (1.76 ± 0.28). Conclusion: The present in vitro study concludes tetracycline HCl root-conditioning agent to be significantly more efficient in smear layer removal, exposing collagen fibers and amount of patent dentinal tubules than doxycycline HCl and EDTA. Clinical significance: The modification of the root surface of human teeth with the use of root conditioning agents leads to enhanced attachment by connective tissue resulting in improved reconstructive periodontal treatment goals.