In this chapter, the microbial population dynamics associated with space environment, influence of spaceflight on the microbial changes, and “omics” approach to elucidate the microbial diversity by using state-of-the-art molecular techniques are discussed. The International Space Station (ISS) is a built environment that has been specially designed to meet its physical characteristics and habitation requirements. The microgravity, exposure to space radiation, and increased carbon dioxide concentrations are conditions that are unique to the ISS. Furthermore, a large proportion of the space within the ISS environment is occupied by astronauts. Learning the makeup of the microbial community within this closed system will improve safety and maintenance procedures. The approach seeks to characterize the viable microbiome of the built environment of the ISS when molecular techniques are used and then to determine if the built environments of cleanrooms on Earth designed for space exploration are a suitable simulation of the ISS environment.