Mushroom survey and collection were conducted in the Dong Hua Sao National Biodiversity Conservation Area (DHSNBCA), which is located about 30 km east of Pakse and about 10 km south of the town of Paksong, Champasak Province of Lao-PDR, from from at the early July to October in 2019. During this periods, mushroom specimens were collected from 6 different locations, and then they were identified and classified into 103 species, 74 genera, 37 families, 15 orders and 6 classes by morphological and molecular analyses. The mushrooms belongs to Ascomycota were classified into 10 species, 6 genera, 4 families, 3 orders and 2 classes, while those belongs to Basidiomycota were classified into 92 species, 67 genera, 32 families, 11 orders, and 3 classes, respectively. In addition, 1 species of Myxomycota was also identified. Among these mushrooms, the most species-rich families are Marasmiaceae (17.3%), Polyporaceae (13.1%), Mycenaceae (8.9%), Agricaceae (8.3%), Xylariaceae (6.5%), Auriculariaceae (4.8%), Ganodermtaceae (3.6%), Dacrymycetaceae (3.0%), Meruliaceae (3.0%), Russulaceae (3.0%) and comprised 71.4% of the total specimens identified.