Abstract Background Human-designed oligotrophic environments, such as cleanrooms, harbor unique microbial communities shaped by selective pressures like temperature, humidity, nutrient availability, cleaning reagents, and radiation. Maintaining the biological cleanliness of NASA’s mission-associated cleanrooms, where spacecraft are assembled and tested, is critical for planetary protection. Even with stringent controls such as regulated airflow, temperature management, and rigorous cleaning, resilient microorganisms can persist in these environments, posing potential risks for space missions. Results During the Phoenix spacecraft mission, genomes of 215 bacterial isolates were sequenced and based on overall genome-related indices, 53 strains belonging to 26 novel species were recognized. Metagenome mapping indicated less than 0.1% of the reads associated with novel species, suggesting their rarity. Genes responsible for biofilm formation, such as BolA (COG0271) and CvpA (COG1286), were predominantly found in proteobacterial members but were absent in other non-spore-forming and spore-forming species. YqgA (COG1811) was detected in most spore-forming members but was absent in Paenibacillus and non-spore-forming species. Cell fate regulators, COG1774 (YaaT), COG3679 (YlbF, YheA/YmcA), and COG4550 (YmcA, YheA/YmcA), controlling sporulation, competence, and biofilm development processes, were observed in all spore-formers but were missing in non-spore-forming species. COG analyses further revealed resistance-conferring proteins in all spore-formers ( n = 13 species) and eight actinobacterial species, responsible for enhanced membrane transport and signaling under radiation (COG3253), transcription regulation under radiation stress (COG1108), and DNA repair and stress responses (COG2318). Additional functional analysis revealed that Agrococcus phoenicis , Microbacterium canaveralium , and Microbacterium jpeli contained biosynthetic gene clusters (BGCs) for ε-poly-L-lysine, beneficial in food preservation and biomedical applications. Two novel Sphingomonas species exhibited for zeaxanthin, an antioxidant beneficial for eye health. Paenibacillus canaveralius harbored genes for bacillibactin, crucial for iron acquisition. Georgenia phoenicis had BGCs for alkylresorcinols, compounds with antimicrobial and anticancer properties used in food preservation and pharmaceuticals. Conclusion Despite stringent decontamination and controlled environmental conditions, cleanrooms harbor unique bacterial species that form biofilms, resist various stressors, and produce valuable biotechnological compounds. The reduced microbial competition in these environments enhances the discovery of novel microbial diversity, contributing to the mitigation of microbial contamination and fostering biotechnological innovation.