Accurate and informative microbiological testing is essential for guiding diagnosis and management of pneumonia in patients who are critically ill. Sampling of tracheal aspirate (TA) is less invasive compared with mini-bronchoalveolar lavage (mBAL) and is now recommended as a frontline diagnostic approach in patients who are mechanically ventilated, despite the historical belief that TA was suboptimal due to contamination from oral microbes. Advancements in metagenomic next-generation sequencing (mNGS) now permit assessment of airway microbiota without a need for culture and, as such, provide an opportunity to examine differences between mBAL and TA at a resolution previously unachievable. Here, we engaged shotgun mNGS to assess quantitatively the airway microbiome in matched mBAL and TA specimens from a prospective cohort of critically ill adults. We observed moderate differences between sample types across all subjects; however, we found significant compositional similarity in subjects with bacterial pneumonia, whose microbial communities were characterized by dominant pathogens. In contrast, in patients with noninfectious acute respiratory illnesses, significant differences were observed between sample types. Our findings suggest that TA sampling provides a similar assessment of airway microbiota as more invasive testing by mBAL in patients with pneumonia.