Although subclinical mastitis is associated with increased HIV load in breast milk, it is not known whether empirical treatment with antibiotics will reduce breast milk HIV load. We examined the effect of antibiotic treatment for subclinical mastitis on HIV load in breast milk. Seventy-five HIV-infected post-partum women in Malawi with subclinical mastitis were treated with oral amoxicillin/clavulanic acid and were followed between 1 and 24 weeks later. Breastmilk HIV-1 load and sodium concentration were measured and microbiological studies were performed at presentation. At 1 week (n = 34), the proportion of women with elevated breast milk leukocyte counts decreased significantly to 41.2% (p < 0.0001) and there was a nonsignificant increase in breast milk HIV-1 RNA load (p = 0.9264) and sodium concentration (p = 0.08) in the affected breast. At 4 to 12 weeks (n = 63), breast milk HIV-1 RNA load and sodium concentration decreased significantly (p < 0.05) and 17.5% had elevated breast milk leukocyte counts. Treatment with amoxicillin/clavulanic acid was associated with a significant decrease in inflammation of the breast, but breast milk HIV load remained elevated despite a significant decrease from baseline. These findings have important implications regarding how mothers should be counselled on safety of resuming breastfeeding after resolution of subclinical mastitis.