Nowadays, preparing a natural, non-toxic, low-cost, and biodegradable substrate called “Green sheet” is one of the new approaches to Nano sciences and biotechnology. Green sheets have multiple functions in various industries such as food packaging, preparation of wound dressings, hospital clothes, drug delivery systems, perfumery, antiperspirant, antimicrobial, and insect repellent, and they have found a special position. One of their unique applications is the use of cultural heritage, such as conservation in the restoration of ancient works, and historical documents, packaging and transportation of historical objects, and making traditional dolls and embroidery. In this study, a natural sheet was prepared using unreelable cocoons through degumming, opening, and finishing processes in the presence of a natural crosslinking agent and the well-known pad-dry-cure method. Tea tree and oregano plant oils were applied to achieve an antimicrobial property. The antimicrobial activity was assessed against Staphylococcus aureus, Escherichia coli, and Candida albicans through qualitative and quantitative methods. The results showed that the silk sheet had good resistance against the mentioned microorganisms.