The beneficial effects of plant essential oils (EOs) have been known for a long time and now they are used in various fields such as medicine and aromatherapy as well as in food and pharmaceutical industries. In the last few years, the use of plant EOs has been noticed against biodeterioration of cultural heritage, which represents a powerful resource of green conservation strategies. This research aims to evaluate the antimicrobial effect of 3 essential oils of rosemary, eucalyptus and thyme on 5 fungal strains, including Aspergillus niger, Penicillium polonicum, Cladosporium limoniform, Penicillium chrysogenom, Parengyodontium album, and 3 bacterial strains including Bacillus cereus, Bacillus licheniformis, Kocuria rosea isolated from deteriorative areas of Jamé Mosque of Isfahan. The results showed that among the used EOs, thyme EO had the greatest antimicrobial effect on the selected microorganisms. This EO in 50% dilution prevented the growth of the tested microorganisms. Eucalyptus EO showed the lowest antimicrobial effect. This EO did not show any antimicrobial effect on Penicillium chrysogenum. However, eucalyptus EO was effective on the tested bacterial strains with the diameter of non-growth zone of 25, 28 and 15 mm on Bacillus cereus, Bacillus licheniformis and Kocuria rosea, respectively. According to these results, thyme EO can be potentially used to control the biodeterioration of cultural heritage.