BACKGROUND: Potentially pathogenic microorganisms are frequently isolated from tracheostomized children, although evidence for empirical therapy of respiratory exacerbation is limited. We aimed to describe upper airway microbiology as found on endotracheal aspirate (ETA) in tracheostomized children and to correlate it with lower airway microbiology through bronchoalveolar lavage fluid. METHODS: We retrospectively reviewed records and airway microbiology of all tracheostomized children under the follow-up care of Queensland Children's Hospital. Subanalysis was based on ventilatory and multidrug-resistant organism status. Sensitivity and specificity of ETA for predicting Pseudomonas aeruginosa and Staphylococcus aureus lower airway isolation were calculated using concomitant bronchoalveolar lavage fluid culture as the accepted standard. RESULTS: From 43 children (18 female, median [interquartile range (IQR)] age 68 (41-115) months, 14 ventilated), 15 different potentially pathogenic microorganisms were isolated (mean 6 SD: 3.30 6 2.23), with S. aureus (n 5 33, 77%) and P. aeruginosa (n 5 29, 67%) predominating. Significantly more types of potentially pathogenic microorganisms were isolated from ventilated children (median 4.00 [IQR 3.25-5.75]) than from nonventilated children (median 2.00 [IQR 1.00-4.00] (P 5 .007), with 93% of ventilated children isolating S. aureus and 86% P. aeruginosa. Multidrug-resistant organisms were present in 12 (28%) children, of whom 8 were ventilated. Methicillin-resistant S. aureus (MRSA) was isolated in 9 (21%) children, of whom 6 were ventilated. For P. aeruginosa and S. aureus isolation, ETA had high sensitivity (95% and 100%, respectively) but low specificity (64.7% and 33.3%, respectively) when compared with bronchoalveolar lavage fluid. CONCLUSIONS: In children with tracheostomy, the predominant respiratory bacterial pathogens were S. aureus and P. aeruginosa, with MRSA being isolated less frequently than previously described. Multidrug-resistant organisms are isolated more frequently from ventilated children. ETA microbiology is a good screening modality, with negative ETA potentially ruling out lower airway S. aureus and P. aeruginosa. Adequately powered prospective studies with quantitative cultures could enhance understanding and guide therapy.