Background Interest in the pulmonary microbiome is growing, particularly in patients undergoing mechanical ventilation. Objectives To explore the pulmonary microbiome over time in patients undergoing prolonged mechanical ventilation and to evaluate the effect of an oral suctioning intervention on the microbiome. Methods This descriptive subanalysis from a clinical trial involved a random sample of 16 participants (7 intervention, 9 control) who received mechanical ventilation for at least 5 days. Five paired oral and tracheal specimens were evaluated for each participant over time. Bacterial DNA from the paired specimens was evaluated using 16S rRNA gene sequencing. Bacterial taxonomy composition, α-diversity (Shannon index), and β-diversity (Morisita-Horn index) were calculated and compared within and between participants. Results Participants were predominantly male (69%) and White (63%), with a mean age of 58 years, and underwent mechanical ventilation for a mean of 9.36 days. Abundant bacterial taxa included Prevotella, Staphylococcus, Streptococcus, Stenotrophomonas , and Veillonella . Mean tracheal α-diversity decreased over time for the total group ( P = .002) and the control group ( P = .02). β-Diversity was lower ( P = .04) in the control group (1.905) than in the intervention group (2.607). Conclusions Prolonged mechanical ventilation was associated with changes in the pulmonary microbiome, with the control group having less diversity. The oral suctioning intervention may have reduced oral-tracheal bacterial transmission.