Abstract The healthcare-associated infections (HCAIs) occur in patients both in nosocomial environments and in community. More often HCAIs are associated to the use of medical devices and bacterial biofilm development on these equipments. Due to the clinical and economic relevance of this topic, new strategies for the treatment of infections caused by biofilm proliferation are unceasingly searched by scientists. The present study investigated the role of vitamin E to reduce the biofilm formation for a larger panel of human pathogens, including strains of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Acinetobacter baumannii, Pseudomonas aeruginosa and Pseudomonas putida. This potential activity was tested by placing a preparation of vitamin E (α-Tocopheryl acetate) as interface between the bacterial culture and the polystyrene walls of a 96 well plate at different concentrations of glucose, used as a biofilm enhancer. The Staphylococcus genus was further investigated by spreading the vitamin E on a silicone catheter lumen and evaluating its influence on the bacterial colonization. From our results, vitamin E has been able to interfere with bacterial biofilm and prevent in vitro biofilm formation. Furthermore, the ability of Staphylococcus aureus and Staphylococcus epidermidis to colonize the catheter surface decreased as a result of vitamin E application.