Etiopathogenesis of acquired and congenital cholesteatoma is still unclear. The clinical behavior of adult acquired, pediatric acquired and congenital cholesteatomas show differences. The scope of the this study was to detect thematrix metalloproteinase(MMP),tissue inhibitors of metalloproteinase(TIMP) andepidermal growth factor receptor(EGFR) gene expression changes in cholesteatoma perimatrix and to compare these changes among congenital cholesteatoma, adult acquired cholesteatoma and pediatric acquired cholesteatoma. A total of 16 genes includingMMPs,TIMPs andEGFRwere analyzed in the samples of 32 cholesteatoma tissues. Real-time PCR was used for detection of the gene expression levels. Data analyses were achieved by Delta Delta CT method (Light Cycler 480 Quantification Software) and Statistical Package for Social Sciences (SPSS) version 22.0. The expression levels ofMMP-2,-9,-10,-11,-13,-14,-15,-16andEGFRgenes were significantly higher in acquired cholesteatoma than healthy tissue (p < 0.05). There was a statistically significant decrease (3.34 times more) in the meanTIMP-2gene expression level in acquired cholesteatoma compared to healthy tissue (p < 0.05). There was a significant increase in the mean expression level ofMMP-7gene and a decrease in the mean expression level ofTIMP-1gene (3.12 times more) in congenital cholesteatoma compared to healthy tissue (p < 0.05). This study indicates that increased expression levels of some particularMMPgenes andEGFRgene and decreased expression levels ofTIMPgenes may play an important role in the development of cholesteatoma. Further,MMP-9,MMP-13andMMP-14genes may have a remarkable role in the development of more aggressive cholesteatoma forms. The authors concluded that overexpression ofMMP-9,MMP-13andMMP-14may cause stronger inflammation associated with cholesteatoma.