The objective of the research study was to determine the content of fatty acids in milk from the Polish Holstein-Friesian cows with regard to the effect of successive lactations and stage of lactation. The research study was performed on 48 selected cows of the Polish Holstein-Friesian breed, at their different ages: lactation I, II, III, and IV (12 cows in each lactation group). From each cow milk samples were taken and the stage of lactation was taken into consideration, i.e. the samples were taken on the following milking days: 6^th, 30^th, 90^th, 300^th, 350^th, and 400^th, (288 samples in total). Among all the fatty acids determined in milk, the contents of the following fatty acids were the highest: C16:0, C18:1 cis 9, C18:0, and C14:0 including those, which are believed to have a potential to reduce the cholesterol level in blood: oleic acid (C18:1 cis 9) and stearic acid (C18:0), and their total content was as high as 37.35 g/100 g of fat. The milk from cows at the lactation stage IV (p ≤ 0.05) and from periparturient cows, i.e. collected on the 6^th day of lactation, was characterized by the highest content of saturated fatty acids (SFA). The content of beneficial unsaturated fatty acids (UFA) decreased in the fat contained in the milk milked at the four successive stages of lactation (p ≤ 0.05) as well as the lactation continued; their content was the highest on the 6^th day and the lowest on the 400^th day 400 (p ≤ 0.01 and p ≤ 0.05). The milk from primiparous cows was characterized by the highest concentration levels of polyunsaturated fatty (PUFA) and monounsaturated fatty (MUFA) acids, and, at the same time, by the lowest content of fatty acids with a less beneficial effect on human health, i.e. C12:0, C14:0, and C16:0. As the lactation continued, the content of MUFA in milk fat decreased and the content of PUFA increased; this fact was confirmed by statistical differences at p ≤ 0.01 and p ≤ 0.05. During a period from the 6^th to the 90^th day of lactation, the ratio of n-6 to n-3 fatty acids was the most advantageous and the contents of functional fatty acids were the highest, i.e. of the butyric (BA), cis 9 oleic (OA), linoleic (LA), short-chain (SCFA), and long-chain (LCFA) fatty acids. The levels of cis 9 trans 11 conjugated linoleic (CLA) and trans 10+11 vaccenic (TVA) acids were the lowest during the lactation yield peak and they increased as the lactation continued to reach the highest values on the 300^th day of lactation.