The aim of this work was to determine the physiological level of neopterin in human breast milk, and to study its variability depending on the duration of a single feeding and the lactation stage. Breast milk samples from 74 women were collected between 2 and 4 days after delivery, and at 15, 30, and 90 days after delivery. Additionally, breast milk samples from eight women were collected before and after 7 and 15 min of breastfeeding. The concentration of neopterin in breast milk was determined by an immunoenzymatic assay. The range of breast milk neopterin concentration at various stages of lactation amounted to 15.4–19.2 nmol/L at 2–4 days after delivery, 20.2–23.0 nmol/L at day 15, 20.8–24.5 nmol/L at day 30, and 16.9–20.4 nmol/L at day 90. The level of neopterin 2–4 days after delivery was significantly lower than that at days 15 and 30; moreover, the concentration of neopterin at day 30 was significantly higher than that at day 90. No significant differences were documented between neopterin concentrations at various phases of a single feeding. While the breast milk concentration of neopterin changes depending on the stage of lactation, it remains stable throughout a single feeding.