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The feasibility of gastrointestinal (GI) microbiome work in a pediatric intensive care unit (PICU) to determine the GI microbiota composition of infants as compared to control infants from the same hospital was investigated. In a single-site observational study at an urban quaternary care children’s hospital in Western Michigan, subjects less than 6 months of age, admitted to the PICU with severe respiratory syncytial virus (RSV) bronchiolitis, were compared to similarly aged control subjects undergoing procedural sedation in the outpatient department. GI microbiome samples were collected at admission (<i>n</i> = 20) and 72 h (<i>n</i> = 19) or at time of sedation (<i>n</i> = 10). GI bacteria were analyzed by sequencing the V4 region of the 16S rRNA gene. Alpha and beta diversity were calculated. Mechanical ventilation was required for the majority (<i>n</i> = 14) of study patients, and antibiotics were given at baseline (<i>n</i> = 8) and 72 h (<i>n</i> = 9). Control subjects’ bacterial communities contained more <i>Porphyromonas</i>, and <i>Prevotella</i> (<i>p</i> = 0.004) than those of PICU infants. The ratio of <i>Prevotella</i> to <i>Bacteroides</i> was greater in the control than the RSV infants (mean ± SD—1.27 ± 0.85 vs. 0.61 ± 0.75: <i>p</i> = 0.03). Bacterial communities of PICU infants were less diverse than those of controls with a loss of potentially protective populations.