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Agarwood is the most expensive non-construction wood product in the world. As a therapeutic agent, agarwood can cure some diseases, but few studies have been carried out on the antagonistic abilities of endophytic fungi associated with agarwood. Agarwood is mainly found in the genus <i>Aquiaria</i>. The objectives of this study are to understand the antimicrobial activities and their potential as biocontrol agents of the endophytic fungi of <i>Aquilaria sinensis</i>. First, fresh samples of <i>A. sinensis</i> were collected from Yunnan and Guangdong Provinces in 2020–2021, and the endophytic fungi were isolated and identified to genus level based on the phylogenetic analyses of the Internal Transcribed Spacer (ITS) region. In this bioassay, 47 endophytic strains were selected to check their bioactivities against three bacterial pathogens viz. <i>Erwinia amylovora, Pseudomonas syringae,</i> and <i>Salmonella enterica</i>; and three fungal pathogens viz. <i>Alternaria alternata, Botrytis cinerea,</i> and <i>Penicillium digitatum</i>. The antibiosis test was carried out by the dual culture assay (10 days), and among the 47 strains selected, 40 strains belong to 18 genera viz. <i>Alternaria</i>, <i>Annulohypoxylon</i>, <i>Aspergillus</i>, <i>Botryosphaeria</i>, <i>Colletotrichum</i>, <i>Corynespora</i>, <i>Curvularia</i>, <i>Daldinia</i>, <i>Diaporthe</i>, <i>Fusarium</i>, <i>Lasiodiplodia</i>, <i>Neofusicoccum</i>, <i>Neopestalotiopsis</i>, <i>Nigrospora</i>, <i>Paracamarosporium</i>, <i>Pseudopithomyces</i>, <i>Trichoderma</i>, <i>Trichosporon</i> and one strain belongs to Xylariaceae had antimicrobial activities. In particular, <i>Lasiodiplodia</i> sp. (YNA-D3) showed the inhibition of all the bacterial and fungal pathogens with a significant inhibition rate. In addition, the strains viz; <i>Curvularia</i> sp. (GDA-3A9), <i>Diaporthe</i> sp. (GDA-2A1), <i>Lasiodiplodia</i> sp. (YNA-D3), <i>Neofusicoccum</i> sp. (YNA-1C3), <i>Nigrospora</i> sp. (GDA-4C1), and <i>Trichoderma</i> sp. (YNA-1C1) showed significant antimicrobial activities and are considered worthy of further studies to identify individual fungal species and their bioactive compounds. This study enriches the diversity of endophytic fungi associated with agarwood, and their potential antagonistic effects against bacterial and fungal pathogens.