Introduction The bacterial content of donated human milk is either endogenous or introduced via contamination. Defining milk bank bacterial content will allow researchers to devise appropriate tests for significant and commonly encountered organisms. Objective A retrospective audit was conducted on data recorded from the Perron Rotary Express Milk Bank, King Edward Memorial Hospital, Subiaco, Western Australia. This aimed to describe the incidence of bacterial species detected in donated human milk and to identify potentially pathogenic bacteria. Material and methods The data comprised of 2890 batches donated by 448 women between 2007 and 2011. Results Coagulase negative Staphylococcus (CoNS) represented the highest prevalence of bacteria in donated milk, isolated from 85.5% of batches (range: 20 to 650,000 CFU/mL) followed by Acinetobacter species in 8.1% of batches (range: 100 to 180,000 CFU/mL). Staphylococcus aureus was the most prevalent potentially pathogenic bacteria in 5% of batches (range: 40 to 100,000 CFU/mL). Conclusion Further investigation is warranted to better define the risks posed by the presence of toxin-producing S. aureus in raw and pasteurized human milk which may allow minimization of risk to the preterm infants. © 2016 Elsevier Ireland Ltd.