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ABSTRACT Hospitals are subject to strict microbial control. Stringent cleaning and confinement measures in hospitals lead to a decrease in microbial diversity, but an increase in resistance genes. Given the rise of antimicrobial resistances and healthcare-associated infections, understanding the hospital microbiome and its resistome is crucial. This study compared the microbiome and resistome at different levels of confinement (CL) within a single hospital. Using amplicon sequencing, shotgun metagenomics, and genome/plasmid reconstruction, we demonstrate that microbial composition differs in a stable way between the CLs and that the most restrictive confinement level CL1 had the lowest microbial but the highest functional diversity. This CL also exhibited a greater abundance of functions related to virulence, disease, defense, and stress response. Comparison of antibiotic resistance also showed differences among CLs in terms of the selection process and specific mechanisms for antimicrobial/antibiotic resistance. The resistances found in the samples of CL1 were mostly mediated via antibiotic efflux pumps and were mainly located on chromosomes, whereas in the other, less restrictive CL antibiotic resistances were more present on plasmids. This could be of particular importance for patient-related areas (CL2), as the potential spread of antibiotic resistances could be a major concern in this area. Our results show that there are differences in the microbiome and resistome even within a single hospital, reflecting room utilization and confinement. Since restrictive confinement selects for resistant microorganisms, strategies are required to deepen our understanding of dynamic processes of microbiome and resistome within hospital environments.IMPORTANCEEffective measures to combat antibiotic resistances and healthcare-associated infections are urgently needed, including optimization of microbial control. However, previous studies have indicated that stringent control can lead to an increase in the resistance capacities of microbiomes on surfaces. This study adds to previous knowledge by focusing on the conditions in a single hospital, resolving the microbiomes and their resistomes in three different confinement levels (CL): operating room, patient-related areas, and non-patient-related areas. We were able to identify stable key taxa; profiled the capacities of taxa, functions, and antimicrobial resistances (AMR); and reconstruct genomes and plasmids in each CL. Our results show that the most restrictive CL indeed had the highest functional diversity, but that resistances were mostly encoded on chromosomes, indicating a lower possibility of resistance spread. However, clever strategies are still required to strike a balance between microbial control and selective pressures in environments where patients need protection.