The benefits of human milk in preterm infants, a population at high risk for developing adverse outcomes for which breast milk is a protective factor, are widely acknowledged. However, preterms' admission in a neonatal intensive care unit (NICU) and newborn's clinical conditions have been described as significant barriers, leading to lower rates of breastfeeding initiation and duration. Healthcare workers play a crucial role in encouraging breastfeeding. We conducted a cross-sectional survey among nurses working in six Italian NICUs, exploring their knowledge and attitude towards breastfeeding. Although the majority of nurses had a specific breastfeeding education, our results show still some variations among answers regarding aspects of breastfeeding support in this setting. Specifically, family-centered care, transition feeding to the breast, and skin-to-skin practice, despite being extensively addressed by the Neo Baby-Friendly Hospital Initiative, are the items that highlighted a range of answers that could result in conflicting information to mothers.Conclusion: By underlining the gaps of knowledge and attitude towards breastfeeding of nurses working in NICUs, this study provides an insight into what needs to be improved, with the aim of promoting higher rates of breastfeeding in the preterm population. What is Known: • Breastfeeding is particularly challenging in the preterm population, despite its universally recognized health benefits. • Improving healthcare professionals' knowledge and attitude towards breastfeeding has been shown to be crucial for promoting breastfeeding in NICUs. What is New: • Our results provide useful insight into nurses' knowledge and attitude towards breastfeeding in NICU settings. • By acknowledging strengths and weaknesses highlighted by this study, tailored strategies could be developed to improve health staff breastfeeding education and support to parents in NICU settings.