Life detection missions to Mars should be as free of microbial organisms as possible to avoid transporting contaminants on spacecraft surfaces. Any microbes that make the trip to Mars or the round-trip back to Earth may compromise our ability to recognize authentic biosignatures from native Mars organisms. Current planetary protection protocols require that spacecraft components must be assembled in cleanrooms that have nearly aseptic conditions, reducing the chance of microbial contamination. The current research studies samples from NASA’s Jet Propulsion Laboratory Spacecraft Assembly Facility, collected from high-traffic floor surfaces and entryways. Microorganisms from these samples were enriched in liquid media based on 50% MgSO4 and 20% NaClO3 brines representative of Mars’ high-salt environment. Through the use of leading-edge molecular genetic techniques, 16S and 18S rRNA sequences provided detailed descriptions of the changing bacterial and fungal communities at timepoints over a six-month period. Bacterial communities were dominated by staphylococci increasing in diversity over time. Fungal communities were dominated by Saccharomycetes decreasing in diversity over six months. Thirty-eight bacterial isolates were collected after six months in the enrichment brines, identified through Sanger sequencing of 16S rRNA, characterized through morphology and biochemical assays, and grown in a variety of salts to measure tolerances. The culture collection contains halotolerant, Gram-positive bacteria found in Staphylococcus, Brevibacterium, Brachybacterium, and Oceanobacillus genera that can survive in a variety of high-salt brines. Those that persisted in the enrichment brines for an extended period may pose the greatest risk to life detection missions as they might be the most likely to survive and proliferate at the nearsurface of Mars. This research informs efforts to protect Mars from microbial contamination that can confound life detection or harm potential native ecosystems.