Analysis of the human milk microbiome is complicated by the presence of a variable quantity of fat. The fat fraction of human milk is typically discarded prior to analysis. It is assumed that all cells are pelleted out of human milk by high speed centrifugation; however, studies of bovine milk have reported that bacteria may remain trapped within the fat fraction. Here, the bacterial DNA profiles of the fat fraction and cell pellet of human milk (n = 10) were analysed. Human and bacterial DNA was consistently recovered from the fat fraction of human milk (average of 12.4% and 32.7%, respectively). Staphylococcus epidermidis was significantly more abundant in the cell pellet compared to the fat fraction (P = 0.038), and three low-abundance species (< 5% relative abundance) were recovered from one fraction only. However, inclusion of fat reduced the efficiency of DNA extraction by 39%. Culture-based methods were used to quantify the distribution of an exogenously added strain of Staphylococcus aureus in human milk fractions. S. aureus was consistently recovered from the fat fraction (average 28.9%). Bacterial DNA profiles generated from skim milk or cell pellets are not representative of the entire human milk microbiome. These data have critical implications for the design of future work in this field.