Objectives: Human milk (HM) is the optimal diet for neonates, but it does not provide enough nutrients for preterm infants. HM fortifiers based on highly processed mature bovine milk (BMFs) are routinely used for preterm infants despite risks of causing gut dysfunction and systemic infection. Gently-processed bovine colostrum as a fortifier (BCF) may better protect against infection and inflammation. We hypothesized that BCF-fortified HM has enhanced antimicrobial activity against pathogens that commonly cause neonatal sepsis, relative to BMF-fortified HM. Methods: Holder-pasteurized HM samples (10 mothers) were aliquoted into 3 fractions: unfortified HM and HM fortified with either BMF or BCF. The samples were analyzed for pH, lactoferrin concentrations, and antimicrobial activities against Staphylococcus epidermidis, Escherichia coli, and Enterococcus faecalis. Results: HM+BCF had a lower pH and higher lactoferrin levels than HM+BMF, with HM being intermediate. Relative to infant formula, HM decreased the growth of S epidermidis, E coli, and E faecalis, with no difference between preterm and term HM. Addition of BMF abolished the antimicrobial effect of HM against S epidermidis and E faecalis but not E coli. By contrast, addition of BCF into HM enhanced antimicrobial activity against S epidermidis and E coli, relative to unfortified HM. HM+BCF was superior to HM+BMF in inhibiting growth of all tested bacteria. Conclusion: BMF fortification decreased whereas BCF fortification enhanced in vitro antimicrobial activity of HM. This effect may partly be derived from the high levels of antimicrobial factors found in BCF, including lactoferrin. BCF may be a better fortifier than BMF for preterm infants.