Aim: This study comparatively evaluated the quantum of fragility zone and nanoleakage formation at tooth-composite resin restoration interfaces following deproteinization of dentin with deproteinization agents and methylene diphosphate (MDP) and methacryloyloxydecyl dihydrogen thiophosphate (MDTP) containing bondings agent using a confocal microscope. Materials and methods: Class V cavities were prepared on the buccal surface of 40 extracted human premolars, which were considered for the present study and were randomly divided into four groups (n = 10) based on the different agents used for deproteinization. Group I: bromelain; group II: α-tocopherol; group III: 5.25% sodium hypochlorite (NaOCl); and group IV (control): MDP and MDTP containing bonding agent. After drying each tooth, a dentin-bonding agent was applied, cured with visible light, and restored with composite resin. The samples were kept in a 50% alcoholic solution of 1% wt rhodamine B fluorescent dye for 24 hours to allow dye penetration. The teeth were then cut into sections parallel to their long axes using a low-speed diamond disc, the specimens were polished with 600-grit silicon carbide paper and viewed under confocal laser scanning microscopy to analyze the dentin adhesive area. Results: The results of the present in vitro comparative study revealed that group IV (control)—exhibited a maximum reduction in nanoleakage (122.42), followed by group III— NaOCl (154.11), group I—bromelain (206.82), and group II—α-tocopherol (182.09). Conclusion: In this present study, a newer adhesive system (MDP, MDTP containing) helps in reducing fragility zone formation and nanoleakage which will significantly increase the longevity of the restoration and decrease the failure rates. Clinical significance: To reduce the failure rate of composite resin restorations by secondary caries formation and to increase the bonding between the tooth and the composite resin to minimize nanoleakage.