To compare the effects of a human breastmilk-derived fortifier on the antibacterial activity of milk obtained from lactating mothers delivering prematurely with the effects of a powdered fortifier on the same milk.Human milk samples were obtained after the first week of postnatal life from 10 lactating mothers, who had delivered prematurely. A bovine milk-based powdered fortifier and a human breastmilk-based frozen fortifier were evaluated. All mothers were healthy and they were not on any medications, although they were taking prenatal vitamins during lactation. The effects of each fortifier on the antimicrobial activity of milk toward Enterobacter sakazaki (ES), Escherichia coli, Clostridium difficile (CD), and Shigella soneii (SS) were evaluated by both the filter paper method and the growth inhibition method.Human milk inhibited the growth of all of the test organisms. This antibacterial activity was almost totally inhibited by the addition of the bovine protein-based human milk fortifier, while it remained unaffected by the addition of the human breastmilk-based fortifier.Breastmilk from women who have delivered preterm has antibacterial activity that can be affected by the addition of bovine-based fortifier, but not by the addition of a human breastmilk-based fortifier.