<strong>Introduction:</strong> <span style="font-family:Verdana;"><span style="font-family:Verdana;"><span style="font-family:Verdana;">Ap</span></span></span><span><span><span style="font-family:""><span style="font-family:Verdana;">propriate hand hygiene reduces healthcare associated infections (HCAI). The present study was an attempt to determine the real knowledge and practices of healthcare professionals of pediatric emergency care unit in Benin. </span><b><span style="font-family:Verdana;">Methods</span></b><span style="font-family:Verdana;">: This was a three-month observational study. First a trained interviewer observed both the premises and the healthcare professionals during their administering treatment, without their knowledge. Second, their level of knowledge about HCAI was examined using a self-administered questionnaire. Finally, bacteria were cultured/measured from their hands before and after hand-washing through swab samples. </span><b><span style="font-family:Verdana;">Outcome: </span></b><span style="font-family:Verdana;">The modes of microorganism transmission were poorly known (21.6%), but knowledge about the 5 moments for hand hygiene was satisfactory (84.1%). Hand hygiene adherence rate was 6.9% in the study population. Simple hand-washing was prevalent (82%</span></span></span></span><span><span><span style="font-family:""><span style="font-family:Verdana;">). The healthcare professionals did not adhere to the recommended duration for hand-washing. The required hand-washing equipment was inadequate, and alcohol-based handrub dispenser was unavailable. Microorganism isolated from healthcare professionals before hand-washing included </span><i><span style="font-family:Verdana;">Klebsiella</span></i><span> <i><span style="font-family:Verdana;">pneumonia</span></i> <i><span style="font-family:Verdana;">and</span></i> <i><span style="font-family:Verdana;">Enterobacter</span></i> <i><span style="font-family:Verdana;">Cloacae</span></i></span><span style="font-family:Verdana;">. After hand-wash