Background: The composition of breast milk (BM) is dynamic and can be influenced by maternal variables that include the diet and nutritional status. This study examined the association between maternal adherence to a Mediterranean diet (MedDiet) and total antioxidant content of BM and infant urine. Methods: We collected 350 BM samples from mothers and urine samples from their infants. The dietary intakes of the mothers were recorded using a validated 65 items-food frequency questionnaire (FFQ). The total antioxidant status of the samples was assessed using the ferric reducing/antioxidant power (FRAP), the 1, 1-diphenyl-2-picrylhydrazyl (DPPH), thiobarbituric acid reactive substances (TBARS), and thiol quantification assays. Milk protein, calcium, and triglyceride (TG) were also determined using standard biochemical kits. Results: Subjects with the highest MedDiet scores were among the women in the highest tertile (T3) for consumption of dietary fiber, fruits, vegetables, nuts, legumes, and seeds, low-fat dairy, whole grains, and the lowest consumption of red meat, sweetened beverages, and sodium, compared to women in the first tertile (T1) with the lowest MedDiet scores. After adjustment for potential confounders, the individuals in the highest tertile for MedDiet score had a significantly higher level of milk DPPH, and infant urinary DPPH than the lowest tertile and had a significantly higher level of milk protein, FRAP and infant urinary FRAP compared to the T2 (P < 0.05). In addition, the mothers in the T3 for the MedDiet pattern had a significantly lower level of milk TG compared to those within the T1 (P < 0.05). Conclusion: Our findings show that a high maternal adherence to a MedDiet can affect BM composition and their infants' urine