The exact causative factor(s) of bone erosion in cholesteatoma are not known. In recent years, the possible role of cytokines has drawn attention. Since the studies on cytokines in cholesteatoma are limited and depend on histopathological methods, the present work approached this subject by biochemical determination of TNF-α lysosomal enzymes, acid phosphatase (total and tartrate resistant), cathepsin B, leucyl aminopeptidase lysozyme together with non-lysosomal enzymes calpain I and II in 50 cholesteatoma samples (epithelial and subepithelial tissues) in comparison with 14 normal skin samples from the external ear canal. The study revealed significantly increased levels of all previous indices in cholesteatoma epithelium and subepithelial tissues compared with healthy skin. The levels of these indices reflected the clinical severity of the disease as reflected by their significant increase in cases with erosion of two or three ossicles, erosion of dural plate, sinus plate and facial canal and more extensive cholesteatoma. It is likely that TNF-α acts both directly by causing bone erosion and indirectly by stimulating the release of lysosomal enzymes. The latter mechanism is supported by the significant correlations observed between TNF-α and lysosomal enzymes. The non-lysosomal enzymes calpain I and II seem to participate in the bone erosion associated with cholesteatoma by their involvement in collagen destruction. Due to the suggested role of TNF-α in bone destruction associated with cholesteatoma the use of anti-inflammatory drugs should be taken into consideration in otitis media to diminish bone destruction. Similarly, antibiotics should be used to prevent the deleterious effects of bacterial endotoxin.