Tracheostomy bypasses physical barriers that decrease microbial access to the lower airway, which can lead to changes to the lung microbiota. Patients often become chronically colonized with potential pathogens. This study described the incidence and prevalence of specific organisms in a 5-y cohort of children with tracheostomy. This retrospective cohort of children aged 0-18 y with tracheostomy identified associations between microbial species and subject characteristics including reason for tracheostomy, gastrostomy tube (G-tube), fundoplication, and ventilator use using chi-square test or Fisher exact test. Of 113 eligible patients, 79 (57% male) met study inclusion criteria. Reasons for tracheostomy included airway obstruction secondary to craniofacial anomalies in 16 children (20%), upper-airway obstruction in 14 subjects (17.3%), neuromuscular disorder in 19 subjects (24%), bronchopulmonary dysplasia with or without pulmonary hypertension in 17 subjects (21%), and congenital heart disease in 13 subjects (16%). Most (69%) used a ventilator for at least 6 h/d; 63% had a G-tube; 41% also had a Nissen fundoplication. Of the 20% with upper-airway obstruction, one third were ventilator dependent, unlike other diagnoses where 57% used a ventilator. Staphylococcus aureus (52/113), Pseudomonas aeruginosa (43/113), and Stenotrophomonas species (39/113) were the most frequently identified bacterial species. Most microbes identified were not associated with subjects underlying diagnoses, ventilator use, or feeding type. However, there was a significant association between upper-airway obstruction and group B Streptococcus species and G-tube with P. aeruginosa. This retrospective single-site descriptive cohort analysis of pediatric subjects with long-term tracheostomy identified trends in microbial prevalence. The presence of specific bacterial strains was more likely to follow individual subject trajectories than sequential appearance of species. P. aeruginosa was associated with G-tube and Streptococcus species with upper-airway obstruction. Ventilator dependence was not associated with specific microbial profiles.