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 high-efficiency particulate arrestance (HEPA) filter element (air) samples were analyzed by traditional cultivation adenosine triphosphate (ATP) and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) assays to estimate viable microbial populations. In addition vacuum cleaner bag components of two cleanrooms at Jet Propulsion Laboratory (JPL Pasadena CA) were examined concurrently. The 16S rRNA gene sequencing based on Illumina platform was used to elucidate the ISS microbial diversity and explore differences between the microbiomes of the ISS and Earth-based cleanrooms. The statistical analyses of these microbiomes show that Actinobacteria Firmicutes and Proteobacteria dominate in the air and surface of the ISS and the cleanroom samples but vary in abundance. While members of Actinobacteria were predominant in the ISS Proteobacteria the least abundant phylum in the ISS dominated the Earth-based cleanrooms. The viable bacterial population (PMA-treated samples) decreased significantly but the treatment did not appear to have an effect on the bacterial composition (diversity) associated with a sampling site. Viable fungal sequences were not retrieved from the ISS HEPA sample where as highest viable fungal diversity was observed in the Earth-based cleanroom (JPL class 100K) debris. The results of this study provided strong evidence of substantial contribution of human skin-associated microorganisms such as Corynebacterium/Propionibacterium (Actinobacteria),not Staphylococcus (Firmicutes) species as the dominant species in the ISS in terms of viable and total bacterial community structure.