Objective: To investigate a possible contribution of handedness to the development of unilateral acquired cholesteatoma. Patients: All patients with unilateral acquired cholesteatoma attending otorhinolaryngology clinic between August 2009 and February 2012 were asked about their handedness. Results: The study group consisted of 237 right-handed and 31 left-handed (11.8%) patients who included 158 male (59%) and 79 female subjects with an age range of 6 to 81 years (mean, 38.6 yr). There were no ambidexters in the present cohort. Overall, unilateral cholesteatoma involved 146 right (54.5%) and 122 left ears (45.5%). Ten of 31 patients (32.3%) with left hand predominance were female subjects. In the unilateral disease, the right ears were affected more frequently in patients with right-hand dominance (59.5%), and the left ears were more frequently affected in the left-handed patients (83.9%); this difference was statistically significant (p < 0.0001). The sex differences in the distribution of cholesteatoma side and handedness were not statistically significant (p = 0.098). Conclusion: The strong relationship has been observed between the patient's handedness, and laterality of the unilateral acquired cholesteatoma may have contribution in the multifactorial process of cholesteatoma formation.