They serve as feeding, breeding and nursery grounds for economically important marine organisms including endangered species. The tropical seagrass Enhalus acoroides was collected from Had Khanom-Mu Ko Thale Tai National Park, southern Thailand. The objectives of this study were to investigate for the presence of endophyte assemblages in E. acoroides, as well as to describe the diversity of endophytes based on LSU, ITS1, 2, 5.8S rDNA sequence analyses. Forty-two fungal assemblages were isolated and identified through molecular data. This resulted in a diversity of fungal groups of Ascomycota (98%) and Basidiomycota (2%). Three major Ascomycota classes including the Sordariomycetes (36%), Eurotiomycetes (33%) and Dothideomycetes (24%) were determined. The predominant ascomycete orders were the Hypocreales, followed by the Eurotiales and the Capnodiales, respectively. Additionally one taxon belonged to the Russulales, Basidiomycota and was possibly mycorrhiza. This study confirms that E. acoroides harbors a wide diversity of fungal endophytes, and provides a baseline for further studies on fungal-host plant interactions.