In an effort to minimize the probability of forward contamination of pristine extraterrestrial environments, the National Aeronautics and Space Administration requires that all US robotic spacecraft undergo assembly, testing and launch operations (ATLO) in controlled clean-room environments. This study examines the impact of ATLO activity on the microbial diversity and overall bioburden contained within the air of the clean-room facility in which the Mars Exploration Rovers (MERs) underwent final preparations for launch. Air samples were collected from several facility locations and traditional culture-based and molecular methodologies were used to measure microbial burden and diversity. Surprisingly, the greatest estimates of airborne bioburden, as derived from ATP content and cultivation assays, were observed prior to the commencement of MER ATLO activities. Furthermore, airborne microbial diversity gradually declined from the initiation of ATLO on through to launch. Proteobacterial sequences were common in 16S rDNA clone libraries. Conspicuously absent were members of the Firmicutes phylum, which includes the genus Bacillus. In previous studies, species of this genus were repeatedly isolated from the surfaces of spacecraft and clean-room assembly facilities. Increased cleaning and maintenance initiated immediately prior to the start of ATLO activity could explain the observed declines in both airborne bioburden and microbial diversity.