Microorganisms, sensitive to the surrounding environment changes, show how the cave environment can be impacted by human activities. Zhijin Cave, featured with the most well-developed karst landform in China, has been open to tourists for more than 30 years. This study explored the microbial diversity in a karst cave and the impacts of tourism activities on the microbial communities and the community structures of bacteria and archaea in three niches in Zhijin Cave, including the mixture of bacteria and cyanobacteria on the rock wall, the aquatic sediments, and the surface sediments, using 16S rRNA high-throughput sequencing technology. It was found that Actinobacteriota and Proteobacteria were the dominant bacteria in the cave and Crenarchaeota and Thermoplasmatota were the dominant archaea. The correlation between microorganisms and environmental variables in the cave showed that archaea were more affected by pH and ORP than bacteria and F − , Cl − , NO 3 − , and SO 4 2− were all positively relevant to the distribution of most bacteria and archaea in the cave. PICRUSt's prediction of microbial functions also indicated that abundance of the bacteria's functions was higher than that of the archaea. The intention of this study was to improve the understanding, development, and protection of microbial resources in caves.