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Tracheobronchial suctioning is a common procedure in intensive care units, but it is essential to observe aseptic measures because of the fragility of patients. The aim of our work was to evaluate the knowledge and practices of nursing staff in terms of hospital hygiene during tracheobronchial suctioning in an anesthesia-intensive care unit. We conducted a descriptive evaluative study during the period from February 21 to April 30, 2023, and included all healthcare professionals responsible for tracheobronchial suctioning in the anesthesia-intensive care department. Our study revealed gaps in the knowledge and practices of healthcare staff during tracheobronchial suctioning in the anesthesia-intensive care department. A specific care protocol is needed to remedy these problems and ensure patient safety.