Objectives/HypothesisTo determine the effect of repeated reprocessing of pediatric tracheostomy tubes (TTs) on biofilm formation.Study DesignIn vitro microbiological study.MethodsPediatric, uncuffed, polyvinyl chloride (PVC) TTs from two different manufacturers (Tracoe Mini and Shiley) were reprocessed mechanically with household detergent and soaked in sodium hypochlorite (bleach). Two TTs of each brand were reprocessed 0 (control), 10, or 20 times. Twenty 2‐mm coupons were then obtained from each TT, immersed in human mucus, and cultured with either Staphylococcus aureus or Pseudomonas aeruginosa. Biofilm formation was evaluated with bacterial counts.ResultsBacterial counts of S. aureus for both brands were significantly higher on the TTs that were reprocessed 20 times compared to those that were not reprocessed (Tracoe: P = .040, Shiley: P < .0001) or those that were reprocessed 10 times (Tracoe: P = .022, Shiley: P = .0002). There was no difference between controls and TTs reprocessed 10 times (Tracoe: P = .76, Shiley: P = .24). P. aeuruginosa counts were not significantly different among the varying numbers of reprocessing cycles for either Tracoe or Shiley TTs (P = .08 and P = .97, respectively).ConclusionsRepeated reprocessing of PVC TTs with detergent and bleach paradoxically promotes S. aureus biofilm development, possibly due to degradation of the tube surface that facilitates bacterial attachment. Further investigation is needed to determine the optimal technique and limits of reprocessing TTs in clinical practice.Level of EvidenceNA Laryngoscope, 126:996–999, 2016