The Regenerative Enclosed Life Support Module Simulator (REMS) was designed to simulate the conditions aboard the International Space Station (ISS). This unique terrestrial, encapsulated environment for humans and their associated organisms allowed investigations into the microbial communities within an enclosed habitat system, primarily with respect to diversity, phylogeny and the possible impact on human health. To assess time- and/or condition-dependent changes in microbial diversity within REMS, a total of 27 air samples were collected during three consecutive months. The microbial burden and diversity were elucidated using culture-dependent and culture-independent molecular methods. The results indicate that during controlled conditions the total microbial burden detected by culture-dependent techniques (below a detectable level to 102 cells m−3 of air) and intracellular ATP assay was significantly low (102–103 cells m−3 of air), but increased during the uncontrolled post-operation phase (∼104 cells m−3 of air). Only Gram-positive and α-proteobacteria grew under tested culture conditions, with a predominant occurrence of Methylobacterium radiotolerans, and Sphingomonas yanoikuyae. Direct DNA extraction and 16S rDNA sequencing methodology revealed a broader diversity of microbes present in the REMS air (51 species). Unlike culture-dependent analysis, both Gram-positive and proteobacteria were equally represented, while members of a few proteobaterial groups dominated (Rhodopseudomonas, Sphingomonas, Acidovorax, Ralstonia, Acinetobacter, Pseudomonas, and Psychrobacter). Although the presence of several opportunistic pathogens warrants further investigation, the results demonstrated that routine maintenance such as controlling the humidity, crew’s daily cleaning, and air filtration were effective in reducing the microbial burden in the REMS.