Background/Objectives: Docosahexaenoic acid (DHA) is an essential component of breast milk for infant neurocognitive and visual development. Because DHA depends largely on dietary intake, women with low seafood consumption are at increased risk of inadequate DHA contents in breast milk. This study aims to quantify the DHA content in breast milk and assess how recommended dietary supplementation during lactation affects these contents among lactating women in three Balkan countries. Methods: This cross-sectional multicenter study included 128 lactating women (18–35 years). Dietary intake of DHA was assessed using a modified Food Frequency Questionnaire. DHA in breast milk was measured by gas chromatography following standard protocols. Both the FFQ and breast milk samples were collected at two time points: 20–40 (visit 1—V1) and 80–100 days (visit2—V2) postpartum. Results: In lactating women, seafood intake was low (Visit 1: 37.5 (0.0–93.8) g/week), meeting about 28% of the recommended amount, with some variation between countries. Consequently, the mean dietary n-3 PUFA and DHA intake for the total sample was less than 50% and 5% of the recommended daily allowance, respectively. Correlation analysis indicates that DHA content in breast milk is related to dietary DHA intake (Visit 1: r = 0.258, p = 0.001; Visit 2: r = 0.238, p = 0.012). DHA contents failed to reach the 0.3% clinical threshold for optimal neurodevelopment, with average levels ranging from 0.149 to 0.165%. Furthermore, no significant differences were observed between supplemented and unsupplemented women at either time point, highlighting a persistent regional deficit regardless of supplementation status. Conclusions: Lactating women in the Balkan region have a low dietary intake of n-3 PUFA and DHA, which is reflected in the breast milk lipid profile, regardless of standard supplementation. These findings highlight the need for a targeted nutritional strategy both via nutrition and supplementation to improve DHA intake in lactating women, but more importantly in infants.
