Psychosocial stress affects the relationship between prolactin (PRL) and cortisol (CORT). The dynamics of PRL and CORT changes under stress in human milk (HM) are largely unknown. We investigated how maternal stress related to recent life changes affects milk CORT and PRL concentrations. The study involved 116 mothers exclusively breastfeeding 5-month-old infants. Maternal psychological stress was evaluated using the Recent Life Changes Questionnaire (RLCQ). Stress response was determined by administering the cold pressor test and measuring CORT in saliva taken during and in milk collected after the test. Hormones concentrations were assayed using the ELISA method. The hierarchical regression models were run to test the association between maternal RLCQ, salivary CORT, and PRL, and CORT in milk. Maternal RLCQ correlated positively with the CORT in saliva, however, no direct association was found between RLCQ and PRL. After controlling for covariates, a positive association was found between salivary and milk CORT. A negative relationship was observed between salivary CORT and milk PRL. The results of the present study indicate that maternal psychological stress may affect the relationship between CORT and PRL in HM. In response to psychological stress, both hormones transported via milk can program infant development in the early postnatal period.