The coastal marine ecosystems of Vietnam are one of the global biodiversity hotspots, but the biodiversity of marine fungi is not well known. To fill this major gap of knowledge, we assessed the genetic diversity (ITS sequence) of 75 fungal strains isolated from 11 surface coastal marine and deeper waters in Nha Trang Bay and Van Phong Bay using a culturedependent approach and 5 OTUs (Operational Taxonomic Units) of fungi in three representative sampling sites using next-generation sequencing. The results from both approaches shared similar fungal taxonomy to the most abundant phylum (Ascomycota), genera (Candida and Aspergillus) and species (Candida blankii) but were different at less common taxa. Culturable fungal strains in this study belong to 3 phyla, 5 subdivisions, 7 classes, 12 orders, 17 families, 22 genera and at least 40 species, of which 29 species have been identified and several species are likely novel. Among identified species, 12 and 28 are new records in global and Vietnamese marine areas, respectively. The analysis of enzyme activity and the checklist of trophic mode and guild assignment provided valuable additional biological information and suggested the ecological function of planktonic fungi in the marine food web. This is the largest dataset of marine fungal biodiversity on morphology, phylogeny and enzyme activity in the tropical coastal ecosystems of Vietnam and Southeast Asia. Biogeographic aspects, ecological factors and human impact may structure mycoplankton communities in such aquatic habitats.