Microorganisms contribute diverse and fundamental roles in biogeochemical processes. In a given microbial community, individuals interact with one another to form complex regulatory networks in which gatekeepers contribute disproportional roles in sustaining stability, dynamics, and function. Owing to the ecological and functional importance of microbial gatekeeper, this review provides an overview on its history, identification, roles, application in biological sciences, and clinical diagnostics. The roles of microbial gatekeepers can be beneficial or detrimental, which depends on our purpose. As the field is rather new, some limitations are raised, and further efforts devoted to solving these concerns are proposed. Collectively, gatekeepers provide promising targets for sustaining and re-establishing a desired microbial community. However, substantial obstacles, such as factors governing gatekeepers, must be overcome to manipulate gatekeepers as positive guys.