The evidence that breastfeeding reduces mortality and short and long-term morbidity among premature and small babies is well established but breastfeeding rates in neonatal units in the UK remain low. The aim of this study was to develop and evaluate an eLearning module that addresses the learning needs of neonatal clinicians to support breastfeeding on Neonatal Intensive Care Units (NICU). The module focussed on the knowledge areas of anatomy and physiology of lactation and expression. Mixed methodology was used to evaluate the eLearning module and inform its iterative development. This consisted of quasi-experimental pre-test/post-test studies using The Neonatal Unit Clinical Assessment Tool (NUCAT), an on-line objective knowledge test with self ratings of confidence to test the effects of the eLearning module on knowledge, confidence in knowledge and confidence in practice. Semi-structured interviews explored neonatal clinicians’ experiences of undertaking the eLearning module and their perceptions of the feasibility and applicability of the eLearning module as well as their opinions and experiences of breastfeeding support and training. In total 101 neonatal clinicians, including neonatal nurses, doctors, Advanced Neonatal Nurse Practitioners (ANNPs), nursery nurses and students undertook the initial NUCAT assessment of knowledge and confidence. A further 90 clinicians went on to complete the training and post intervention assessment, 60 repeated the post intervention assessment at 6-8 weeks. Baseline knowledge was greater in the area of breast milk expression than in the anatomy and physiology of lactation. Neonatal nurses were found to have greater baseline knowledge of breast milk expression than doctors or nursery nurses. Doctors/ANNPs were more knowledgeable about the anatomy and physiology of lactation. Following the training intervention doctors/ANNPs showed the greatest improvement in knowledge scores and nursery nurses the least. Knowledge and confidence was significantly increased immediately following the intervention and at 6-8 weeks in all groups. The semi-structured interviews conducted after the study showed the feasibility and applicability of the eLearning module for clinicians in NICU. On-line assessment and training provide a potentially effective multidisciplinary training method to improve breastfeeding knowledge and confidence. Nursery nurse may have differing learning needs and require further support and training.