(1) Background: Adequate protein intake plays an essential role in growth and neurodevelopment, especially in preterm infants. We investigated the effects of maternal diet and body mass index (BMI) on human milk (HM) composition. (2) Methods: HM samples were obtained from 136 lactating mothers (BMI: 18.0–36.7 kg/m2), of which 93% gave birth to preterm infants. Macronutrient content in HM was measured by mid-infrared transmission spectroscopy. Leptin and adiponectin were analyzed using appropriate ELISAs. Maternal diet was determined by 24-h recall. (3) Results: Significant positive associations were found between protein, fat, carbohydrate and energy intake, and levels of corresponding macronutrients in HM, especially in protein concentrations (p < 0.001). An increased protein intake was positively correlated with adiponectin (p < 0.001) and leptin (p = 0.035) in HM. Maternal BMI was positively associated with a higher protein level in HM (p < 0.05), as well as with a higher dietary protein intake (p < 0.05). (4) Conclusions: Knowledge of maternal diet and BMI impacting HM composition is essential to optimize the feeding of newborn infants. This is especially relevant in the nutritional management of preterm infants; it can be utilized in approaches to improve growth rates and the appropriate development of infants and to prevent obesity.