We conducted a prospective observational study of tracheostomy tubes with three primary goals: (1) to identify the presence and location of bacterial biofilms on adult tubes, (2) to determine how soon after insertion of a tube the presence of a biofilm could be identified, and (3) to identify the bacterial organisms that formed the biofilms on these tubes. Our study materials consisted of 7 adult tracheostomy tubes that had been changed during a routine outpatient clinic visit or hospital consultation. The tubes were examined for the presence of biofilms on the posterior aspect of the outer cannula; also, specimens were obtained from the posterosuperior aspect of the cuff on the 3 tubes that had a cuff. Samples of 2 to 3 mm were taken from each site and analyzed by scanning electron microscopy. Bacterial biofilms were found on 4 of the 7 tubes; they were present on the outer cannula of 3 cuffless tubes that had been inserted 14 days, 4 months, and 2 years previously and on the cuff of 1 tube that had been inserted 10 days previously. The biofilms were composed of gram-positive cocci in pairs that were likely consistent with Staphylococcus epidermidis.