UNLABELLED: In microbiota research, whole stool sampling is the conventional approach but can be problematic or infeasible for certain patients. This study aims to validate the use of rectal swabbing as an alternative method for microbiota analysis and determine optimal storage conditions suitable for various clinical settings, including intensive care units. We evaluated different sampling techniques and storage temperatures. Our findings indicated that rectal swabs yield microbiota diversity comparable to whole stool samples. Notably, storage conditions significantly impacted microbiota profiles, with increased IMPORTANCE: We developed a pragmatic approach to study total and aerobic gut microbiota, applicable in numerous clinical units, such as intensive care or emergency units, where whole stool sampling is often impractical. This approach employs ESwab devices, which are already commonly used in hospitals.