This study was carried out to assess the effect of using pasture combined with total mixed ration (TMR) on milk production and composition, fatty acid (FA) profiles, fat-soluble antioxidant content, and total milk antioxidant capacity (TAC). In addition, the effect of milk pasteurization and storage at 2 °C for 4 days, lipophilic antioxidants and TAC were assessed. Two homogeneous groups of Jersey cows (n = 8) were constituted, which were randomly assigned to one of the two treatments: TMR (without access to pasture), or partial mixed diet (pTMR: grazing 8 h/day + TMR indoors). To establish FA profiles and lipophilic antioxidants’ changes in milk during the grazing period, in case of the pTMR group the experimental period was spilt in three grazing periods: P1 (May), P2 (June), and P3 (June/July). Milk yielded from cows having limited access on pasture (pTMR diet) showed an improved FA profile, with higher concentrations of FAs considered benefic for human health (vaccenic acid (VA), conjugated linoleic acid (CLA), omega-3 FA (n-3 FA)) (p < 0.01) and a lower concentration of FAs with hypercholesterolemiant potential (C12:0, C14:0, and C16:0) (p < 0.05), compared to that of the TMR diet. This change in FA profile was correlated with improved sanogenous lipid indices of milk fat (n-6/n-3 FA atherogenic index and thrombogenic index). Milk yielded during the P1 grazing period had higher concentrations of VA, CLA, and n-3 FA (p < 0.05) and lower concentrations of C14:0 and C16:0 (p < 0.01); it exhibited the best values for the main sanogenous fat lipid indices of fat. Moreover, pTMR milk showed a higher α-tocopherol, retinol, and β-carotene content (p < 0.05), positively correlated with TAC values in milk (P1 ˃ P2 ˃ P3). By comparison, cows fed using the TMR diet yielded a higher quantity of milk (p < 0.05), but a lower fat and protein content (p < 0.01), and also a higher saturated FAs and n-6 FA content (p < 0.05) together with a lower concentration of lipophilic antioxidants in milk. Thermal treatment showed no effect on α-tocopherol and retinol content in milk, but their concentrations decreased during the storage, at the same time a TAC decrease. The results of this study prove the positive effect of using pasture combined with TMR on FA profiles, milk antioxidant content, and antioxidant capacity, with beneficial effects on nutrition and health in humans.