The aim of this study was to determine the concentrations of alpha- and gamma-tocopherols in human breast milk samples from different periods of lactation and to compare them with tocopherol content in commercially available formulas for infants at corresponding ages. The study included 93 breast milk samples obtained on the 2nd (colostrum, n = 17), 14th (n = 30), 30th (n = 27) and 90th day of lactation (n = 19), along with 90 samples of commercially available initial and follow-on infant formulas. Concentrations of tocopherols were determined using normal-phase high-performance liquid chromatography. Depending on the stage of lactation, human breast milk contained 2.07–9.99 mg L−1 of alpha-tocopherol and 0.22–0.60 mg L−1 of gamma-tocopherol. Breast milk concentrations of alpha-tocopherol decreased with the time of lactation, while significant differences in gamma-tocopherol concentration were observed only between the 14th and 30th day of lactation. There was no significant correlation between the dietary intake of vitamin E and its estimated breast milk concentration, also in women who declared vitamin supplementation. Compared with colostrum, infant formulas were characterised by significantly lower concentrations of alpha-tocopherol and vitamin E. This finding indicates the need of additional vitamin E supplementation of bottle-fed infants during the initial 2–3 days of life.