The interaction between environmental factors and the distribution of gobiid fishes was investigated at 16 sampling sites in estuarine areas of the Mekong Delta, Vietnam. Fish specimens were mainly collected by using trawl net, push net, and bag net every two months, from August 2017 to August 2019. Data analysis showed that 40 fish species belong to three families in the gobiid assemblage in the studied area. The most common family was Gobiidae (62%), followed by Oxudercidae (25%) and Eleotridae (13%). The number of fish species decreased gradually from estuary to midstream of river systems. The gobiid assemblage was more diversified in the river system controlled by the West Sea (37 species and 696 individuals) than in the river planned by the East Sea (17 species and 225 individuals). The fish species and biodiversity indexes decreased with the reduction of salinity from the estuary to the midstream of the river system. In the wet season, the fish assemblage was more abundant and diverse than in the dry season due to the environmental factors influenced by the Mekong River. The highest biodiversity index was recorded in Bay Hap and Ong Trang (77.38%). Moreover, the analyzed result of biodiversity indexes revealed that the diversity of fish species in the study area was relatively high. The study outcome will be useful in fisheries management and biodiversity conservation, especially for estuaries.