Most donor human milk (HM) banks use Holder pasteurization (HoP) to ensure microbiological safety, although it can degrade essential bioactive factors for newborns. This study evaluates the innovative ultra-high-pressure homogenization (UHPH) technology as a potential alternative. Listeria innocua, Staphylococcus carnosus, Franconibacter helveticus (formerly named Cronobacter helveticus) and Escherichia coli strains were used as surrogates for common HM pathogens according to European Milk Bank Association (EMBA) guidelines, to evaluate the efficacy of new technologies. A reconstituted powder milk formula inoculated with these strains was used to determine the most efficient conditions (those to achieve a lethality of ≥5 Log), applying treatments from 150 to 300 MPa. These treatments were later validated using inoculated HM with the same strains. Immunoglobulin (sIgA, IgG, IgM) retention was also evaluated and compared with HoP. Results showed that UHPH treatments at 200 MPa achieved a lethality > 5 Log for all strains, except for St. carnosus, which required 250 MPa for complete inactivation in HM. Unlike HoP, UHPH at 200 and 250 MPa did not significantly reduce the basal concentration of sIgA, IgG, or IgM compared with raw HM. These findings suggest UHPH as a promising alternative to HoP, maintaining both microbiological safety and immunological quality.