ABSTRACT Objective: To evaluate the relationship between the maternal diet quality and the fatty acid composition of breast milk in the first trimester of lactation. Methods: This is an observational cross-sectional epidemiological study of nursing mothers. The data collection instruments were a semi-structured questionnaire for sample characterization and a recall of usual intake. Diet quality was assessed based on the healthy eating index (HEI). Samples of mature breast milk were obtained by hand milking. Milk fat was extracted using the Bligh-Dyer method and methylated with 0.25 mol/L sodium methoxide in methanol diethyl ether. A gas chromatograph equipped with a flame ionization detector determined the milk fatty acid profile. Pearson’s and Spearman’s correlation tests evaluated association between the variables. Subsequently, a multiple linear regression model was built and multivariate regression analysis was applied. Results: Our findings revealed an inverse relationship between the consumption of total fruits and the polyunsaturated fatty acid profile and a direct association of the intake of total fruits and total grains with monounsaturated and saturated fatty acids. Conclusions: The results of this study suggest that maternal diet quality affects the fatty acid composition of breast milk.