OBJECTIVE To investigate a possible contribution of handedness to the development of unilateral acquired cholesteatoma. METHODS 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). CONCLUSIONS 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.