Background: Human milk banks have a pivotal role in provide optimal food for those infants who are not fully breastfeed, by allowing human milk from donors to be collected, processed and appropriately distributed. Donor human milk (DHM) is usually preserved by Holder pasteurization, considered to be the gold standard to ensure the microbiology safety and nutritional value of milk. However, as stated by the European Milk Banking Association (EMBA) there is a need to implement the improvement of the operating procedure of human milk banks including preserving and storing techniques. Aim: The purpose of this study was to assess the effectiveness and safety of the selected new combination of methods for preserving donor human milk in comparison with thermal treatment (Holder pasteurization). Methods: We assessed (1) the concentration of bioactive components (insulin, adiponectin, leptin, activity of pancreatic lipase, and hepatocyte growth factor) and (2) microbiological safety in raw and pasteurized, high-pressure processed and lyophilization human breast milk. Results: The combination of two techniques, high-pressure processing and freeze-drying, showed the best potential for preserving the nutritional value of human milk and were evaluated for microbiological safety. Microbiological safety assessment excluded the possibility of using freeze-drying alone for human milk sample preservation. However, it can be used as a method for long-term storage of milk samples, which have previously been preserved via other processes. Conclusion: The results show that high-pressure treatment is the best method for preservation that ensures microbiological safety and biological activity but subsequent freeze -drying allowed long-term storage without loss of properties.