Objective To compare the histopathological structure and immunohistochemical characteristics of acquired cholesteatoma in children and adults. Study design Prospective clinical study in a tertiary care centers. Material & methods This study was conducted on 40 patients presenting with cholesteatomatous middle ear disease. Twenty patients were of a pediatric age group (<18years) and the rest were adults (>18years). Patients were admitted to either the ENT Department of Alexandria University Hospital or that of the Students’ Hospital. All cholesteatoma specimens were collected intra-operatively and preserved for histopathological examination and immunohistochemical technique (PCNA monoclonal antibody). Results Histopathological examination of the submitted specimens revealed that strips of stratified squamous epithelium with the underlying tissues were fibrous in adults while cellular inflammatory infiltrates were seen in children. The degree of fibrosis was significantly higher in the adult group. Immunohistochemical examination revealed significantly higher expression scores of the proliferation marker in the pediatric group. In the matrix: The mean of PCNA/LI of adult squamous epithelium was 17.75±12.08 and of children’s cases was 32.82±17.09. In the perimatrix: The mean of PCNA/LI of adult inflammatory infiltrate was 14.77±17.28 and in children was 24.22±22.61. The differences of both were statistically significant. Conclusion The degree of fibrosis was significantly higher in adult specimens indicating a reparative process and less invasiveness. PCNA expression was significantly higher in the matrix and perimatrix of pediatric cholesteatoma as correlated to its increased proliferation. Pediatric cholesteatoma was more aggressive and invasive than adult cholesteatoma.