Purpose <p>Pediatric tracheostomy tubes (TT) are frequently colonized by bacteria and biofilm, and evidence regarding optimal cleaning methods remains limited, particularly in the context of recent global shortages encouraging tube reuse. The primary aim of this study was to evaluate the effectiveness of a standardized cleaning protocol for pediatric TT by comparing microbiological cultures obtained before and after cleaning. Secondary objectives included characterizing TT microbiology and exploring the use of a dental plaque disclosing solution as a qualitative marker of biofilm presence.</p> Methods <p>A before-and-after study was conducted in 23 pediatric patients with tracheostomies at a tertiary children’s hospital. TT were cultured from internal and external surfaces prior to cleaning and again after undergoing a standardized cleaning protocol using water, dishwashing detergent, and mechanical brushing. Biofilm presence was qualitatively assessed using a dental plaque disclosing solution. Pre- and post-cleaning culture results were compared using McNemar’s test.</p> Results <p>Before cleaning, 95.5% of TT demonstrated positive bacterial cultures, most commonly <i>Pseudomonas aeruginosa</i> (60.9%). After cleaning, all external surface cultures were negative, and internal lumen cultures were negative in 95.8% of cases (<i>p</i> &lt; 0.05). Visible staining with the disclosing solution was present in all TT prior to cleaning and absent in all cases after cleaning.</p> Conclusion <p>A standardized cleaning protocol using water, detergent, and mechanical brushing was effective in significantly reducing bacterial colonization and eliminating visible biofilm from pediatric TT. Dental plaque disclosing solution may represent a useful qualitative tool for assessing biofilm presence.</p>