Introduction: Surface treatment of avulsed tooth must be directed towards the debridement of necrotic periodontal fibers while having no effect on the cementum microhardness. The role of proteolytic enzymes on the periodontal ligament (PDL) surface and hardness of cementum has not been investigated in the literature. Aim: The present study aimed to evaluate the effect of 5.25% sodium hypochlorite (NaOCl), 10% bromelain, 10% papain and 10% panzyme on root surface topography and microhardness of avulsed teeth. Materials and Methods: Forty healthy premolars were freshly extracted and randomly allocated into five treatment groups based on the surface agent employed; Group 1: 50 ml saline, Group 2:20 ml of 5.25% NaOCl, Group 3: 20 ml of 10% bromelain enzyme, Group 4: 20 ml of 10% papain enzyme, and Group 5: 20 ml of 10% mixed panzyme enzyme. The specimens were then subjected to confocal laser scanning microscopy and scanning electron microscopic examination. Supplemental set of freshly extracted forty premolars were decoronated longitudinally and one-half from each specimen was submitted for Vickers microhardness testing before and after application of the above-mentioned agents. Statistical Analysis: Intragroup comparison of mean percentage hardness reduction was assessed using paired t-test. Results: Treatment with NaOCl led to complete removal of PDL fibers with the presence of cracks and crazes on the root surface along with a significant reduction in microhardness. Bromelain and papain revealed regular and even distribution, while few PDL fibers were evident with papain. Panzyme revealed a shiny surface with minute cracks and a significant reduction in microhardness. Conclusion: Ten percentage bromelain for 10 min was effective in the removal of necrotic PDL fibers and preserved the cementum integrity better.