We analysed knowledge of and adherence to guidelines for the prevention of catheter-related infection (CRI) among Spanish healthcare workers (HCWs) from paediatric and neonatal intensive care units by distributing 357 questionnaires to 31 Spanish hospitals. The overall mean scores for individual knowledge and daily practice were 5.61 and 5.78, respectively. Our results reveal room for improvement in Spanish HCWs' knowledge of prevention of CRI. Continuing education programmes and implementation of care bundles must be introduced to improve prevention and management of CRI.