Mastitis is a common inflammatory breast condition that affects approximately 20% of Australian lactating women. Although the pathogenesis of mastitis remains unclear, Staphylococcus aureus is thought to have a role in many cases. S. aureus is more frequently isolated from the milk of women with mastitis compared to women without mastitis and this bacterium has a wide range of virulence factors that would allow it to cause damage and inflammation in the milk ducts. However, S. aureus has also been identified more frequently from milk with lower lactose concentrations, such as early postpartum milk, which share biochemical characteristics with mastitis milk. The aim of this study was to investigate whether the presence of S. aureus in human milk is associated with milk lactose concentration. Human milk samples were aseptically collected from mothers participating in the Drakenstein Child Health Study (Cape Town, South Africa) at a single time point between 5-10 weeks postpartum. These samples underwent Nuclear Magnetic Resonance spectroscopy to determine their metabolome, and a subset of these samples (n=117) were cultured to isolate S. aureus. A binomial generalised linear model and chi-squared tests were used to examine the association between the presence of S. aureus and the lactose concentration in the human milk samples. The lactose concentrations in these samples ranged between 39.1mM and 283.5mM (mean concentration of 210.9mM). Samples with lactose concentrations below 166mM were considered “low lactose” based on the Iglewicz and Hoaglin outlier test. The presence of S. aureus in human milk was strongly associated with the “low lactose” milk phenotype (OR 13.61, 95% CI: 5.25-38.94). Low lactose concentrations in human milk may be more permissive for the growth of S. aureus and may therefore be a potential risk factor for the development of S. aureus-associated mastitis.