This study aims to identify the optimal conditions-host, plant material, seasonality, and agricultural practices-for isolating and developing a collection of culturable endophytic microorganisms to support sustainable L. cultivation. Samples were collected from three Sicilian olive cultivars ('Nocellara del Belice', 'Nocellara Etnea', and 'Nocellara Messinese') and six wild olive accessions across different phenological phases and under organic and conventional agronomic management. Endophytes were isolated from leaves and twigs using a culture-dependent approach, and their taxonomic diversity and plant-growth-promoting (PGP) traits were analyzed. A total of 133 endophytic isolates were identified, spanning bacterial (, , and ) and fungal ( and ) phyla. Wild olive trees contributed more than cultivated varieties to enriching the diversity and composition of culturable endophyte collection as well as twigs instead of leaves. Winter sampling allowed to implement the taxonomic genera of olive endophyte collection. Both farming systems favored an increase in the composition of microbial collection, though organic farming systems supported greater microbial richness. Functional analysis highlighted key PGP traits in a selection of bacterial isolates, including indole-3-acetic acid and siderophore production, nitrogen fixation, and antifungal activity. spp. dominated enzymatic activities, such as amylase, protease, and lipase production, as well as antifungal activity against the olive fungal pathogen . This research highlights the significant diversity and functional potential of Mediterranean olive endophytes. Our findings emphasize the role of native microbial communities as bio-inoculants, promoting plant growth, nutrient uptake, and disease resistance. These insights lay the groundwork for developing targeted olive-microbial consortia for biocontrol and stress tolerance applications.