Proteases are involved in the pathogenicity of allergy, increasing epithelial permeability and acting as adjuvants. Enzymatic activity is therefore important for the allergenicity of an extract and also affects its stability and safety. However, the enzymatic activity of extracts is not usually evaluated. The objective of this study was to evaluate the enzymatic activity of the most allergenic mite extracts and to investigate their allergenic properties. Extracts from nine allergenic mite species (Dermatophagoides pteronyssinus, Dermatophagoides farinae Hughes, Euroglyphus maynei, Lepidoglyphus destructor, Tyrophagus putrescentiae (Schrank), Glycyphagus domesticus (DeGeer), Acarus siro L., Chortoglyphus arcuatus, and Blomia tropicalis) were characterized. Protein and allergen profiles were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western-blot, respectively. Gelatinolytic activity was evaluated with a zymogram and the activity of other enzymes (cysteine, serine proteases, and esterases) was evaluated individually or with the API-ZYM system. The main differences in protease activity were found between house dust mites and storage mites. House dust mites presented higher cysteine protease activity while storage mites presented higher serine protease activity. These differences are in line with their trophic specialization. A wide range of different activities was found in all the extracts analyzed, reflecting the fact that the extracts preserve the activity of many enzymes, this being necessary for a correct diagnosis. However, enzymes may act as adjuvants and, therefore, could lead to undesirable effects in immunotherapies, making this activity not suitable for treatment products. Modified extracts with lower enzymatic activity could be more appropriate for immunotherapy.