Aim This study explored the attitudes of medical professionals to organ and tissue donation in paediatric intensive care units (PICUs) and neonatal intensive care units (NICUs) in the Netherlands. It also examined their compliance with the existing Dutch donation protocol and assessed whether a paediatric donation protocol was needed. Methods We invited 966 professionals working in all eight PICUs and the two largest NICUs to complete an online survey from December 2016 until April 2017. Results A quarter (25%) took part and they included PICU intensivists, neonatologists, nurses and other health and allied professionals. Most were female and nurses. More than half (54%) of the PICU respondents considered paediatric organ donation to be very important and 53% supported tissue donation. In contrast, only 22% of the NICU respondents believed that both neonatal organ and tissue donation were very important. Familiarity and compliance with the existing national donation protocol were low. PICU nurses had significantly less experience than PICU intensivists and felt less comfortable with the donation process. None of the NICU respondents had prior donation experience. Conclusion Paediatric intensive care units and NICU professionals lack specialised knowledge and experience on organ and tissue donation. A comprehensive and clear paediatric donation protocol is clearly needed.