Objectives <p>Ventilator-associated pneumonia (VAP) is a frequent and serious infection among mechanically ventilated children in pediatric intensive care units (PICUs), with oral colonization playing a key role in its pathogenesis. Effective oral hygiene measures are therefore crucial to reduce VAP incidence and improve clinical outcomes. This study aimed to compare the incidence of VAP in PICU patients receiving routine oral care versus toothbrushing with chlorhexidine, and to evaluate the impact on mechanical ventilation duration, PICU stay, and mortality with secondary outcomes including microbiological colonization, inflammatory markers, and inotropic support requirements.</p> Design <p>A parallel-group superiority randomized controlled trial.</p> Methods <p>A randomized controlled trial was conducted on 118 children aged 18 months to 16 years, including PICU patients requiring invasive mechanical ventilation for ≥ 48&#xa0;h. On admission, patients were randomly allocated into two groups: the intervention group received oral care with toothbrushing plus 0.12% chlorhexidine mouth rinse, while the control group received routine care with 0.12% chlorhexidine rinse only. Both procedures were performed three times daily. VAP was diagnosed according to CDC criteria. Quantitative and qualitative analyses assessed variables distinguishing VAP cases from controls, and multivariate logistic regression identified independent predictors of disease severity.</p> Results <p>The results of our study revealed that VAP occurred in 31 patients (52.5%) in the control group and 25 patients (42.3%) in the intervention group, indicating a lower incidence with toothbrushing plus chlorhexidine, although this difference was not statistically significant (<i>p</i> = 0.36). Among all participants, 45.8% died and 54.2% were discharged. Mortality was higher in the control group (53.7%) compared with the intervention group (46.3%), while discharge rates favored the intervention group (53.1% vs. 46.9%); however, these differences did not reach statistical significance (<i>p</i> = 0.46). Positive sputum cultures were more frequent in the control group (55.6%) than in the intervention group (44.4%), whereas negative cultures were more common among intervention patients (54.7% vs. 45.3%), with no significant difference between groups (<i>p</i> = 0.268).</p> Conclusion <p>Although adding toothbrushing to routine chlorhexidine oral care did not produce a statistically significant reduction in VAP incidence or mortality, a consistent trend toward clinical benefit was observed across multiple outcome measures, including lower rates of positive sputum cultures, reduced inotropic support requirements, and fewer new pulmonary infiltrates. These findings highlight the potential value of enhanced oral hygiene in the PICU and support the need for larger multicenter studies to confirm its effect.</p> Trial registration <p>ClinicalTrials.gov, NCT07287566. Registered on November 20, 2025. Retrospectively registered at <a href="https://clinicaltrials.gov/ct2/show/NCT07287566">https://clinicaltrials.gov/ct2/show/NCT07287566</a>.</p> Protocol and statistical analysis plan access <p>The full study protocol and the statistical analysis plan are included in this document and are available from the corresponding author upon reasonable request.</p>