Background <p>During mechanical ventilation, the inflated cuff of endotracheal tubes (ETTs) may help to minimise patient secretions containing microorganisms from reaching the lower trachea and distal ETT surface. The primary study aim was to determine if there were differences in biofilm formation above and below the cuff using microbial community profiling.</p> Methods <p>In this prospective observational study, ETTs were collected following extubation of mechanically ventilated critically ill adults. The biofilm community composition and structure on ETT segments were examined by culture-independent (Illumina MiSeq 16S&#xa0;rRNA marker-gene sequencing) and -dependent (extended-quantitative culture) methods and related to clinical variables.</p> Results <p>Sixty-four participants were recruited following a median (IQR) of 3.77 (1.56–7.49) days intubation. Although participant-level differences in communities were observed, most had evidence of multispecies biofilm development on the ETT surface. Community profiling indicated that there was no difference in alpha- and beta-diversity metrics or density in paired segments from above and below the ETT cuff. Culture-independent analysis detected a greater biofilm richness and more often identified microorganisms reported with clinically-directed culture of respiratory secretions than extended-quantitative culture (82.35% vs. 52.94%). At extubation a higher community density was associated with increased richness (<i>p</i> = 0.01) and diversity (<i>p</i> = 0.03) and reduced evenness (<i>p</i> = 0.01) and dominance (<i>p</i> = 0.03), but not bacterial pathogen or yeast presence/abundance by culture. Participant characteristics were not associated with the extent of biofilm community density.</p> Conclusions <p>Biofilm communities forming above and below the ETT cuff are highly individual but conserved suggesting that an inflated ETT cuff does not provide an effective microbial barrier.</p>