Background : The aim of this study was to compare the differential Ki-67 and p27 staining properties of acquired cholesteatoma in adult patients for prognostic analysis. Methods: Forty-two adult patients with acquired cholesteatoma were enrolled. The cholesteatoma and matched meatal skin tissues of the patients were immunostained with Ki-67 and p27 antibodies. Canal wall down mastoidectomy was performed in all patients. The differential staining properties––positive staining in the cholesteatoma and negative staining in the skin tissue (C+S-), negative staining in the cholesteatoma and positive staining in the skin tissue(C-S+)––were compared for bone erosion scores (BES), stage, and recurrence rates. Results: Isolated findings in the cholesteatoma tissues, without matching with the skin tissues, demonstrated that stage and recurrence rates were not related to findings in the cholesteatoma tissues ( P ˃ .05). However, C+S- for Ki-67 and C-S+ for p27 are risk factors for worse prognosis including advanced stage ( P < .001 for Ki-67 and P = .008 for p27), BES values ( P < .001 for Ki-67 and P = .001 for p27), and recurrence rates ( P < .001 for Ki-67 and P = .037 for p27). Conclusion: This is the first paper assessing the cholesteatoma prognosis according to the differential Ki-67 and p27 staining properties of cholesteatoma and healthy skin tissues. Cellular proliferation rate in the cholesteatoma is important but insufficient by itself for predicting the prognosis of cholesteatoma patients. Patients having lower basal levels of cellular proliferation rate and higher cellular activity in the cholesteatoma tissue are prone to worse prognosis with increased stage, recurrence rates, and degree of bone erosion.