Agarwood is broadly used in incense and medicine. Traditionally, agarwood formation is induced by wounding the trunks and branches of some species of Aquilaria spp., including A. sinensis. As recently evidenced, some fungi or their fermentation liquid may have the potential of inducing agarwood formation. The present study aimed to analyze the fungi isolated from an agarwood-producing A. sinensis tree and subsequently identify the fungi capable of promoting agarwood formation. We identified a total of 110 fungi isolates based on their morphological characteristics and rDNA ITS sequences. These isolates came from four different layers (namely the decomposing layer, agarwood layer, transition layer, and normal layer) near the agarwood formation site of the trunk. According to the experimental results, most of them belonged to Dothideomycetes (81.82%), while the others to Sordariomycetes (13.64%) or Eurotiomycetes (4.55%). Of note, 88 isolates were shown belonging to the species of Lasiodiplodia theobromae that are most frequently isolated from different layers. In addition, when the fermentation liquid of two isolates of L. theobromae (AF4 and AF12) and one isolate of Fusarium solani (AF21) was inoculated into the A. sinensis wood using the Agar-Wit technique, promoted agarwood formation was observed; however, the effect of AF21 did not keep stable in the later test, while AF4 and AF12 still functioned 1 year later. This study may lay a foundation for exploring the underlying mechanism of agarwood formation as well as fungi application in agarwood production.