Fatty acids are some of the most important components of human milk. Polyunsaturated fatty acids (PUFAs) are essential nutrients required for the optimal growth and development of infants, especially the central nervous system, brain and retina. To determine the conjugated linolenic acid (CLA) and vaccenic acid (VA) content of human breast milk from mothers consuming different diets, and to compare the results with CLA and VA levels in infant formulas (IF) and follow-up formulas (FF). Fifty healthy mothers were classified according to their diet status into one of two groups: diet low in dairy products and conventional diet without limiting the intake of dairy products. Dietary intake of dairy fat was determined based on 3-day food diaries. Fatty acid (FA) composition in samples were analyzed by High Resolution Gas Chromatography (HR-GC). In the group of 20 mothers whose diets were deficient in dairy products, the average CLA content of breast milk fat was determined to be 0.27% of total FA, the VA 0.36%. In the group of 30 women consuming dairy products, the average content of CLA and VA in breast milk fat was statistically significantly higher: 0.49% and 0.69% of total FAs, respectively. In the fat of the IF and FF tested (n=11) only trace amounts of both FA were found. The results of the study indicate that CLA and VA concentrations of human milk can be influenced by diet. It is recommended that the source of these FAs in the diet of breastfeeding women are natural products and not dietary supplements. The majority of commercially available IF and FF do not contain sufficient amounts of CLA and VA, and that their FA composition is deficient in comparison with breast milk fat.