The aim of the study was to assess the influence of cow breed and feeding season on the concentration of health-promoting, bioactive peptides in cows’ milk. Milk from Holstein-Friesian cows (HF) contained more bioactive peptides than milk from Simmental cows (SIM), regardless of season, including peptides with specific biological functions. HF milk showed higher concentrations of colostrinin (HF: 413.65 ng/ml; SIM: 371.14 ng/ml), casokinin (HF: 90.48 ng/ml; SIM: 68.89 ng/ml), casomorphin (HF: 87.73 μmol/ml; SIM: 76.17 μmol/ml), and ceruloplasmin (HF: 2141.63 ng/ml; SIM: 1547.94 ng/ml). These results indicate stronger neuroprotective, anticancer, antioxidant, immunomodulatory, and anti-obesity properties of HF milk. Milk collected in summer, particularly from cows fed silage and maize silage, showed more favourable characteristics. It contained higher concentrations of health-related components, including copeptin (summer: 467.84 ng/ml; winter: 406.26 ng/ml). Increased levels of bioactive substances in summer milk are likely attributable to a greater share of forage, especially silage, and a reduced proportion of legume feed. However, it was also observed that increasing the percentage of starchrich feed, such as cereals and maize silage, negatively affected the biological value of milk and reduced the levels of biologically active peptides.