Levels of fatty acid (FA) in breast milk (BM) may vary depending on the maternal diet. This study aimed to explore FA composition in BM of lactating women following dietary restrictions due to infant allergic conditions. Thai lactating mothers of term infants who were on exclusion diets were recruited. Mature BM was collected before and after a period (at least 2 weeks) of dietary restriction. FA in BM was analyzed by gas chromatography-mass spectrometry. Fifty lactating women 33.7 ± 3.6 years of age were enrolled. Thirty-three percent of the lactating mothers restricted more than eight food items. Most common dietary restriction were cow's milk (88%) and eggs (74%). After the period of dietary exclusion, total polyunsaturated FA showed no significant change, while saturated FA (SFA) declined, and monounsaturated FA (MUFA) increased. A decrease in fat intake was associated with an increase in arachidonic acid (ARA) and docosahexaenoic acid (DHA) content in BM (r = -0.37, r = -0.36; p < 0.05). However, a rise in ARA, eicosapentaenoic acid (EPA), and DHA intake was associated with an increase in linoleic acid and EPA in BM, respectively (r = 0.38, r = 0.55 and r = 0.41; p < 0.05). Infant weight-for-age z-score did not significantly change after the period of maternal dietary exclusion. Maternal exclusion diet resulted in lower SFA and higher MUFA composition in BM. Further study should explore the long-term outcomes of maternal dietary restriction on infant and child health.