The aim of this study was to evaluate iron and zinc concentrations in the mature human milk (HM) and to investigate the relationship between these concentrations and maternal factors. HM samples were collected between 4–6 weeks postpartum from 32 healthy, exclusively breastfeeding mothers. The assessment of dietary intake during breastfeeding was based on a food frequency questionnaire and three-day dietary records. Nutritional status of participants was assessed with body mass index and body composition analysis, measured with bioelectrical impedance. HM intake was assessed with infants’ weighting, whereas iron and zinc contents in HM were determined by inductively coupled plasma mass spectrometer. The median intake of HM was 492.5 mL (466–528.5) and the concentrations of HM iron and zinc were 0.33 mg/L (0.26–0.46) and 2.12 mg/L (1.97–2.45), respectively. Maternal total zinc and iron intake (diet + supplementation) was positively correlated with their concentrations in HM. Consumption frequency of meat, vegetables and legumes was revealed to be a significant factor influencing zinc concentration in HM. Regarding iron, it was the consumption frequency of meat, fish and seafood, vegetables and legumes, nuts and seeds. The intake of iron from HM was low, and after assuming a mean fractional iron absorption, it was only 0.038 mg/d. Our results show that maternal diet influences iron and zinc content in HM, suggesting that adequate intake of food rich in investigated minerals may be a positive factor for their concentrations in HM.