SCOPE Necrotizing enterocolitis is a serious and life-threatening condition affecting very low birth weight neonates. The composition of human milk oligosaccharides in breast milk varies significantly. This nested case-control study aimed to evaluate the association between levels of the HMO 2-fucosyllactose in breast milk and the occurrence and severity of NEC. METHODS AND RESULTS A total of 247 mother-infant dyads, between 26 and 32 weeks of gestation, were enrolled over a one-year period in a nested case-control study. Breast milk samples were collected between the 9th and 15th day of life. Clinical data for enrolled infants were reviewed at discharge or at six weeks of life. Each case was matched with five controls (1:5). Only matched samples were analyzed for 2’-fucosyllactose (2-FL) levels using LC-MS/MS. The median 2-FL levels in cases were significantly lower compared to controls (p < 0.03). However, univariate analysis did not show a significant association. An inverse correlation was observed between NEC severity and 2’-FL levels. Notably, 2’-FL levels below 311 µg/mL demonstrated a sensitivity of 80% in predicting NEC occurrence. 2. CONCLUSION Studies are needed to explore the role of 2’-FL in NEC prevention and its potential as a supplement.