Abstract Background: In order to ascertain any bacterial contamination of donor human milk, we cultured milk samples both before and after Holder pasteurization. Methods: During 2003, all frozen donor milk samples were thawed and aseptically placed in pools prior to pasteurization. All individual and pooled samples were cultured and incubated for 48 hours. Specific microbiologic identification of group B Streptococcus , Staphylococcus aureus , coagulase-negative Staphylococcus , α - Streptococcus , Enterococcus , and Bacillus sp. was performed. Gram-negative rods (GNRs) were subtyped but not specifically identified. Positive cultures were expressed in quantitative colony counts. Results: Samples from 303 pools and 810 individual mother's donor milk were cultured. Forty-four pools (15%) represented “preterm donor milk” from milk donated by mothers who delivered prior to 36 weeks of gestation. Before pasteurization, 185 pools (62%) grew at least one lactose-fermenting GNR, and 58 pools (19%) grew a second lactose-fermenting GNR. Forty-four percent grew a non–lactose-fermenting GNR, and 20% grew an oxidase-positive GNR. The majority (87%) were colonized with Staphylococcus . Other Gram-positive organisms identified included Enterococcus ( 16%), α - Streptococcus (8%), and S. aureus (4%). Only one milk sample was positive for methicillin-resistant S. aureus . The majority of pooled milk samples grew two or more organisms. Milk from 22% of mothers did not grow detectable bacteria on routine cultures. The bacteriologic colonization profiles of milk from mothers delivering prematurely were similar to those of mothers delivering at term. After routine Holder pasteurization of donor milk, 93% of milk samples showed no growth on routine bacterial cultures. Conclusions: A wide variety of bacteria contaminate human milk from donor mothers. Most milk samples grew coagulase-negative Staphylococcus and Gram-negative organisms. Although normal skin flora was cultured typically from milk samples, there were also many prepasteurization milk samples contaminated with pathogenic organisms. Holder pasteurization was, however, an effective means by which to remove any detectable bacteria from samples of donor human milk.