Background Human health is closely interconnected with its microbiome. Resilient microbiomes in, on and around the human body will be key for safe and successful long-term space travel. However, longitudinal dynamics of microbiomes inside confined built environments are still poorly understood. Herein we used the Hawaii Space Exploration Analog and Simulation IV (HI-SEAS IV) mission, a one year-long isolation study, to investigate microbial transfer between crew and habitat, in order to understand adverse developments which may occur in a future outpost on the Moon or Mars. Results Longitudinal 16S rRNA gene profiles, as well as quantitative observations, revealed significant differences in microbial diversity, abundance and composition between samples of the built environment and its crew. The microbiome composition and diversity associated with abiotic surfaces was found to be rather stable, whereas the microbial skin profiles of individual crewmembers were highly dynamic, resulting in an increased microbiome diversity at the end of the isolation period. The skin microbiome dynamics were especially pronounced by a regular transfer of the indicator species Methanobrevibacter between crewmembers within the first 200 days. Quantitative information was used to track the propagation of antimicrobial resistance in the habitat. Together with functional and phenotypic predictions, quantitative and qualitative data supported the observation of a delayed longitudinal microbial homogenization between crew and habitat surfaces which was mainly caused by a malfunctioning sanitary facility. Conclusions This study highlights main routes of microbial transfer, interaction of the crew and origins of microbial dynamics in an isolated environment. We identify key targets of microbial monitoring, and emphasize the need for defined baselines of microbiome diversity and abundance on surfaces and crew skin. Targeted manipulation to counteract adverse developments of the microbiome could be a highly important strategy to ensure safety during future space endeavors.