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A wide array of bacteria and fungi are known for their association with pests that impact the health of the olive tree. The latter presents the most economically important cultivation in Tunisia. The microbial diversity associated with olive orchards in Tunisia remains unknown and undetermined. This study investigated microbial diversity to elucidate the microbial interactions that lead to olive disease, and the bio-prospects for potential microbial biocontrol agents associated with insect pests of economic relevance for olive cultivation in the Mediterranean area. Bacterial and fungal isolation was made from soil and olive tree pests. A total of 215 bacterial and fungal strains were randomly isolated from eight different biotopes situated in Sfax (Tunisia), with different management practices. 16S rRNA and ITS gene sequencing were used to identify the microbial community. The majority of the isolated bacteria, Staphylococcus, Bacillus, Alcaligenes, and Providencia, are typical of the olive ecosystem and the most common fungi are Penicillium, Aspergillus, and Cladosporium. The different olive orchards depicted distinct communities, and exhibited dissimilar amounts of bacteria and fungi with distinct ecological functions that could be considered as promising resources in biological control.