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The disinfection procedures aim to reduce the microbial load, but there are doubts about the risks of contamination spreading into the lumens of devices, such as three-way stopcocks (3-WS). This study aimed at an in vitro evaluation of the antibacterial procedure of manual friction of 3-WS intentionally contaminated and to determine the solution dispersion into the lumens. Laboratory experiments were developed in two steps: evaluation of bacterial spread through intentional contamination with Staphylococcus aureus and Pseudomonas aeruginosa, and alcohol dispersion into the 3-WS lumens. After manual friction of the 3-WS with saline solution at 0.85% (w/v) [control group], S. aureus and P. aeruginosa were isolated in the lumens of 55.6% and 27.8% of the devices, respectively. However, after the disinfection of the 3-WS with ethyl alcohol at 70% (w/v), there was no bacterial contamination in the lumens of the 3-WS. On the other hand, the solution dispersion (dye) into the lumens was evidenced by two different techniques: Durham tubes (5.6%) and swabs (46.3%). The manual friction of the 3-WS with ethyl alcohol at 70% demonstrated antibacterial efficacy, but it refers to reflections on the risk of solution diffusion into the venous network and the inherent clinical practice situations and patient safety.