An audit was carried out in paediatric wards to study the compliance of healthcare workers (HCWs) to the procedures recommended for the control of hospital-acquired diarrhoea. Thirty-two paediatric wards in the southeast of France participated on a voluntary basis in this prospective observational study after completing a self-administered questionnaire recording measures of hygiene. All the observations were made by the same investigator and focused on preventive procedures: use of single room, handwashing, hand disinfection, overclothing, single-use gloves and masks. Two hundred and seventy patient–HCW contacts were observed, including mainly diapering, temperature measurement, collection of blood sample and catheter care. The isolation of patients in a single room and use of gowns by HCWs were significantly associated with diarrhoea. Whereas handwashing before care was performed by HCWs in more than 95% of all the procedures, the compliance in the use of disposable gloves by HCWs was only of 39.4% for technical procedures (including those with potential exposure to blood) and 20.3% for diapering or temperature measurement. A substantial agreement between reported and observed measures of hygiene was observed for handwashing before contact and hand disinfection with antimicrobial soap before contact. In contrast, this agreement was moderate for use of single room, handwashing after contact, overclothing and wearing disposable gloves after a diaper change. Despite the excellent compliance of HCWs to handwashing, clearer recommendations for the indication and use of disinfectants and disposable gloves are urgently needed.