Objective - To determine the micronucleus (MN) frequency of acquired cholesteatoma tissue using an MN assay. Material and Methods - Eighteen patients were diagnosed as having chronic otitis media with acquired cholesteatoma and were divided into primary and secondary acquired cholesteatoma groups. Cholesteatoma tissue and normal tissue specimens from the external ear canal skin were taken from the patients during surgical operations. MN frequencies of cholesteatoma and control samples were determined according to standard criteria. Results - The MN frequencies of the cholesteatoma and control tissues were 0.54%±0.31% and 0.24%±0.11%, respectively (p < 0.01). MN frequencies for the primary and secondary acquired cholesteatoma groups were 0.63%±0.36% and 0.46%± 0.26%, respectively (p > 0.05). MN frequencies in cholesteatoma patients without and with complications were 0.42%± 0.19% and 0.85%±0.37%, respectively (p < 0.05). Conclusion - MN frequencies were found to be increased in cholesteatoma tissues when compared with external ear canal skin. The MN frequency in five cases with complications was higher than in cases without complications. These results indicate that there could be associations between MN frequency and acquired cholesteatoma and between MN frequency and complications.