To reveal characteristics of microbial community structure in tunnel type underground reservoir in island areas. By comparing the microbial communities in fully enclosed reservoirs, semi-closed reservoirs, and raw water using high-throughput sequencing of bacterial 16S rRNA and microeukaryotic 18S rRNA genes, we found that the alpha diversity of bacterial communities followed the order: fully enclosed reservoir water > raw water > semi-closed reservoir water. In contrast, the alpha diversity of microeukaryotic communities followed the order: raw water > semi-closed reservoir water > fully enclosed reservoir water. Additionally, the absolute dominant species varied among these water sources. Combined with the correlation analysis between environmental factors and microorganisms, the bacterial community and microeukaryotes community are significantly affected by environmental factors as a whole, and bacterial community is more affected by environmental factors than microeukaryotes community. The composition of microbial community structure in the water body is different under different conditions, whether it is bacteria or microeukaryotes, and bacterial community is more sensitive to environmental factors than microeukaryotes community. Leading to a comprehensive understanding of the characteristics of microbial community under different conditions of tunnel type underground reservoir at the perspective of microbial communities.