Background: Human milk is essential for a child’s best development. However, what a mother eats while breastfeeding can directly influence the composition of mother’s milk. Research Aim: This study aimed to assess the antioxidant-oxidant profile of human milk and establish a connection between this profile and the dietary habits of the mothers. Methods: A cross-sectional study was conducted at the Hospital Infantil e Maternidade Alzir Bernardino Alves (HIMABA), located in the municipality of Vila Velha-ES, Brazil. The sample included 98 participants. All volunteers completed a structured interview and a Food Frequency Questionnaire. Approximately 5–10 ml of colostrum, transitional milk, and mature milk were manually collected. The antioxidant activity of human milk was assessed using the colorimetric method for free radical scavenging with 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid. Oxidative stress was determined by measuring lipid peroxidation through malondialdehyde concentration, evaluating advanced oxidation protein products, and assessing total protein content using the Bradford method. Results: The antioxidant profile of colostrum was higher than that observed in later phases of milk, whereas pro-oxidants increased in later phases. Maternal dietary patterns influenced the pro-oxidant status of human milk. Participants with a higher daily intake of milk, dairy products, vegetable oils, olive oils, and legumes exhibited lower levels of lipid peroxidation in colostrum, transition milk, and mature milk, respectively. Conclusions: Our study highlights the vital role of a balanced maternal diet in shaping the pro-oxidant status of human milk, with implications for infant health.