This study aimed to increase both rumenic acid (RA, cis-9 trans-11 C18:2), the main conjugated linoleic acid (CLA) isomer and α-linolenic content in ewes’ milk fat under field conditions by dietary means and to investigate the effects of these changes on milk processing and Manchego cheese quality. Bulk milk and milk from individual ewes that received a control ration or a ration supplemented with flax seed (1.85 g 100 g−1 of dry matter) and sunflower oil (0.81 g 100 g−1 of dry matter) were collected for a period of 45 days. Milk production and composition were not affected by lipid supplementation but a significant increase (P<0.05) in the milk fat RA and α-linolenic acid concentration was observed. Levels of RA, linoleic and α-linolenic acids increased from 0.47, 0.36 and 1.99 to 0.85, 0.66 and 2.53 g 100 g−1 of milk fat, respectively, with supplemented diet. The ratio of n-3/n-6 changed from 1:6 to 1:4 with lipid addition to the rations. Most of the CLA isomers were increased in enriched milk but the most remarkable increases were observed for 11–13 and 12–14 positional isomers. Overall, sensory characteristics of Manchego cheeses made with enriched milk did not substantially differ from those made with milk from ewes fed with non-supplemented diets.