The safety of the International Space Station (ISS) crewmembers and maintenance of ISS hardware are the primary rationale for monitoring microorganisms in this closed habitat. The composition of the microbial community of this built environment is unique due to microgravity, space radiation, and elevated carbon dioxide levels. As built environments are known to have their own microbiomes, next-generation sequencing methods have to be utilized to explore the ISS microbial profile, and use this data for further development of safety and maintenance practices. ISS vacuum cleaner bag components (surface) and vacuum cleaner bag components of two cleanrooms at Jet Propulsion Laboratory (JPL, Pasadena, CA) were examined. 16S rRNA gene and ITS sequencing based on the 454 platform was used to elucidate the ISS microbial diversity and explore differences between the microbiomes of the ISS and Earth-based cleanrooms.