(1) Background: Premature infants require mothers’ milk fortification to meet nutrition needs, but breast milk composition may be variable leading to a risk of inadequate nutrition. We aimed at determining factors influencing mothers’ milk macronutrients. (2) Methods: Milk samples were analyzed for the first 5 weeks after premature delivery, by infrared spectroscopy. Mothers’ nutritional intake data were obtained during standardized interviews with dieticians then analyzed with reference software. (3) Results: Composition of 367 milk samples from 81 mothers was (Median [range]g/100mL): Carbohydrates 6.8[4.4-7.3], lipids 3.4[1.3-6.4], proteins 1.3[0.1-3.1]. There was a relationship of milk composition with mothers’ carbohydrates intake only (r=0.164; p<.01). Postnatal age was correlated with milk proteins (r=-0.505 p<.001) & carbohydrates (r=+0.202, p<.001). Multiple linear regression analyses showed (coefficient) a relationship between milk proteins r=0.547 and postnatal age (-0.028), carbohydrates intake (+0.449) and the absence of maturation (-0.066); and between milk lipids r=0.295 and carbohydrates intake (+1.279) and smoking (-0.557). Finally, between milk carbohydrates concentration r=0.266 and postnatal age (+0.012) and smoking (-0.167). (4) Conclusions: Variability of mothers’ milk composition is differentially associated for each macronutrient with maternal carbohydrates intake, antenatal steroids, smoking, and postnatal age. Improvement in milk composition could be achieved by modification of these related factors.