Background: Ventilator-associated pneumonia (VAP) is one of the most common causes of nosocomial infections and is associated with prolonged hospitalization, increased health care costs, and high mortality of critically ill patients during hospitalization in intensive care units (ICUs). Objective: To characterize and evaluate in vitro and in vivo antimicrobial and anti-biofilm activity of an in-house tracheostomy tube impregnated with chlorhexidine and violet crystal. Methods: The tracheostomy tubes were tested in vitro for their ability to prevent biofilm formation by standard strains of S. aureus, P. aeruginosa, and E. coli, and multidrug-resistant bacteria obtained from clinical cultures: Meticillin-resistant S. aureus (MRSA), and carbapenem-resistant Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae. Results: The impregnated tracheostomy tubes demonstrated antimicrobial activity, including for multidrug-resistant bacteria. In this pilot study, 14 patients were evaluated, seven in the chlorhexidine and violet crystal-coated group and seven in the control group. During ventilation, VAP occurred in one patient in the coated group and in three patients in the control group (p=0.28). The biomass in the impregnated tubes did not differ from the control group and no difference was found in the production of sessile cells by the quantitative method, with a median of 15.50 cfu/mL (IQR25-75% 12.00-196.50) and 168.00 cfu/mL ((IQR25-75% 78.50-250.00), respectively. Conclusion: This study provides preliminary evidence to support that antiseptic impregnation of tracheostomy tube provides significant antimicrobial activity.