© 2021 Elsevier LtdNatural ingredients as an alternative to chemical additives in the food industry are rapidly becoming widespread due to the increasing consumer concern. Lysozyme and lactoferrin are found in high amounts in donkey milk and are known to have antimicrobial effects on many microorganisms. In this study, we extracted these proteins from donkey milk with lysozyme-imprinted and lactoferrin-imprinted cryogel columns using FPLC and applied them to Kashar cheese as an antimicrobial agent by dipping into a solution. The treated cheeses were compared with control samples for their physicochemical, textural, sensory and microbiological properties. Results demonstrated that products treated with antimicrobial proteins exhibited lower microbial load, better physicochemical, textural and sensorial properties throughout the ripening periods. Results of this study highlight lysozyme and lactoferrin from donkey milk for potential usage as natural antimicrobial agents to prolong the shelf-life of products, provide consumer safety and health and obtain better quality characteristics.