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The Al-Aqsa Mosque (Al-Haram Al-Sharif) is occupying the eastern southern part of the Old City of Jerusalem. The Dome of the Rock, the Dome of the Al-Aqsa Mosque, along with minarets, riwaqs, walls, other small domes and arches and the annex buildings dominate the whole site of the Old City and are essential elements of its skyline. To our knowledge, no studies concerning the microbiota contents and their degradative enzymes of Al-Aqsa mosque exists up till now and they deserve a solid study even it will be considered as a preliminary one due to the hard political situation which made the sampling is very hard mission. The purpose of this study is to explore actinobacterial and fungal communities in different substrates (soil, wood and stones) of unique holy place, Al-Aqsa Mosque, Al-Quds, Palestine and to evaluate their diversity and degradative enzyme potentialities of some selected taxa.