Premature infants require higher nutrient intake to support their growth and development. Consequently, fortifying breast milk has become standard care to meet these infants' nutritional needs. However, the macronutrient composition of breast milk is highly variable among mothers and throughout lactation, leading to challenges in managing nutrient content. Understanding the factors that may affect changes in breast milk macronutrients should be considered. This study aims to identify factors affecting macronutrient levels in breast milk from mothers of preterm infants in Thailand by examining maternal nutrition and demographic factors. A non-experimental correlational study design was employed to investigate how these factors influence macronutrient components in mature milk. Forty-seven mothers who gave premature birth were selected through convenience purposive sampling. Breast milk samples were collected at two-time points: during 1-2 weeks of lactation (time point 1) and 3-4 weeks of lactation (time point 2). Milk samples were analyzed using the MIRIS human milk analyzer (HMA). Maternal dietary intake was assessed using 24-hour dietary food recall records from two non-consecutive days and food frequency questionnaires (FFQs). Maternal body mass index (BMI) was measured using current body weight and height. The findings revealed significant changes in breast milk carbohydrates and protein levels from colostrum and transitional milk to mature milk (carbohydrates: Z = -3.511, p < .001; proteins: Z = -5.155, p < .001). However, fat and energy levels did not differ significantly (fat: Z = -1.96, p = .05; energy: Z = -1.498, p = .134). Multiple linear regression analysis showed a significant positive association between breast milk macronutrients and maternal dietary intake, including daily intake of carbohydrates, fats, and protein. However, no association was found between maternal BMI and breast milk macronutrients. Among maternal demographic factors, only the mode of delivery significantly influenced breast milk macronutrient levels. These findings support that maternal factors, particularly dietary intake, can affect the nutritional value of breast milk. Monitoring and modifying these related factors may improve breast milk macronutrient content for preterm infants.