Disinfection of gloved hands is advocated increasingly in situations where visibly unsoiled gloves are used during multiple clinical activities on the same patient. As there are no data demonstrating that such practice attributes to lower bacterial transfer during clinical care, a standardized experimental study was conducted. Gloved hands touched chicken breasts contaminated with Staphylococcus epidermidis with or without disinfection before touching sterile catheter valves. Contaminated gloves transferred 5.18 log10 colony-forming units (cfu) S. epidermidis to the catheter valves. Disinfection of contaminated gloves significantly reduced the numbers transferred to 0.78 log10 cfu. Disinfection of gloved hands may reduce the risk of transmission.