: Cholesteatoma is a destructive middle ear pathology requiring precise surgical removal to prevent recurrence and preserve auditory function. The chemically assisted dissection (CADISS) system (AuXin Surgery, Ottignies-Louvain-la-Neuve, Belgium), based on Mesna (5%), was introduced to enhance tissue separation and minimize residual disease. : This study aimed to compare the cleaning efficiency of CADISS-assisted dissection versus the conventional manual dissection of cholesteatoma from incus bone surfaces using quantitative ultrastructural analysis. : This retrospective study evaluated 67 human incus samples collected during cholesteatoma surgery-35 treated with manual dissection and 32 with CADISS. Samples were imaged using variable pressure scanning electron microscopy (VP-SEM) in hydrated conditions. Clean area/total area ratios were calculated and analyzed statistically using non-parametric tests. Postoperative MRI follow-up at 1 month was conducted to assess residual disease. : CADISS-assisted samples demonstrated significantly higher clean area/total area ratios (mean: 0.2095 vs. 0.0478, < 0.0001). Qualitative imaging showed fewer residuals > 1 mm in the CADISS group (9% vs. 77%). MRI follow-up revealed a lower recurrence rate in the CADISS group (3.1%) compared to manual dissection (11.4%). : CADISS-assisted dissection provides superior cholesteatoma debris removal compared to manual methods, as evidenced by VP-SEM imaging and clinical follow-up. This technique may improve surgical outcomes and reduce recurrence risk in middle ear surgery.