Automatic flushing of milking clusters between milking events is a control measure aimed at reducing transmission of mastitis pathogens from infectious milk to a subsequently milked cow. We evaluated the effect of flushing with cold water and flushing with water containing peracetic acid (PAA) on the concentration of Staphylococcus aureus in teat cup liners. Thirty-two clusters in a swing-over milking parlor (Dairymaster, Causeway, Ireland) were subjected to a simulated milking with S. aureus–contaminated milk. Sixteen clusters were not flushed (controls), whereas 8 clusters were flushed with cold water (966 ± 32 mL) and 8 clusters were flushed with water containing PAA (200 mL/mL). A random teat cup in each cluster was sampled by rinsing with a phosphate buffer. Teat cup samples were cultured on the day following collection on Baird-Parker plates to determine the concentration of S. aureus. In teat cup samples from control clusters, the mean concentration of S. aureus was 2.8 × 10⁵ cfu/mL. The concentration of S. aureus was zero in teat cup samples from clusters flushed with cold water. In teat cup samples from clusters flushed with water containing PAA, the concentration of S. aureus was in general reduced compared with control clusters, but S. aureus was not removed completely. However, the automatic cluster flushing did not function properly when clusters were flushed with water containing PAA; thus, results reflected the effect of inadequate function rather than the effect of adding disinfectant to the flushing water. Before the main study, we conducted a pilot study to evaluate whether teat cup sampling with swabs and sample analysis with quantitative PCR were appropriate methods for the main study. Specifically, we evaluated the effect of swab sample mass on detection of S. aureus by quantitative PCR in the laboratory, Further, we compared PCR and bacterial culture on detection of S. aureus in a suspension following disinfection of the suspension with PAA. We sampled 20 identical S. aureus suspensions for culture and PCR by swabs before and after disinfection with PAA. Swab sample mass was determined by differential weighing and contributed to 46% of the variation observed in detection of S. aureus by PCR. Following disinfection with PAA, S. aureus remained detectable by PCR, although culturability ceased. Based on these results, we sampled teat cups in the main study with a buffer rinse and quantified S. aureus in the samples by bacterial culture. We concluded that automatic cluster flushing with cold water was effective in removing S. aureus from teat cup liners and that addition of PAA was therefore not necessary.