As the survival rate for preterm infants increases, more emphasis is placed on improving health-related quality of life through optimal nutritional management. Human Milk Banks (HMBs) provide bioactive nutrients and probiotic microorganisms to premature newborns, especially in the first year of life. Donated milk screening and selection of potential donors ensures the quality and microbiological safety of the donated milk. Therefore we reviewed the basic characteristics of donors and the amounts and contamination of breast milk donated to the Regional Human Milk Bank (RHMB) in Warsaw. In four years, the RHMB collected 1445.59 L of milk, of which 96.60% was distributed among hospitalised infants. Additionally, breastmilk from donor candidates (139 samples from 96 women) was tested at least once in the first year of lactation. First analyses showed that 18 women’s milk samples were microbiologically pure, and 78 samples had one or more species of commensal and/or potentially pathogenic bacteria. In human milk samples from 31 women, the bacteria level was above the standard required by the RHMB; therefore, donors were re-educated, and further samples were tested. Most women followed the recommendations on hygienic expression and storage of milk before transfer to the RHMB. Our analysis will help to increase the accessibility and quality of raw donor milk and to meet the needs of more newborns.