Our aim was to evaluate the copy number alterations of chromosomes 3, 7, 8, and 17 in middle ear cholesteatomas and define the association between the rate of cell proliferation and chromosome number changes. Tissues were obtained from 16 patients. Fluorescence in situ hybridization was performed on tumor imprint preparations. Cell proliferation was characterized with Ki-67 monoclonal antibody on cholesteatoma samples and on postauricular skins as control. Different degrees of aneusomy were found for all chromosomes except for chromosome 3. Chromosome copy number alterations were associated with elevated proliferative rate and related also with the aggressiveness of the lesions. Based on our results, we assume that aneusomy of chromosomes 7, 8, and 17 might play an important role during invasion of the adjacent bony structures of cholesteatoma, as well as associate with increased cell proliferation activity, which might lead to the aggressive behavior of the tissue.