BACKGROUND Dental plaque biofilms are colonized by respiratory pathogens in mechanically-ventilated intensive care unit patients. Thus, improvements in oral hygiene in these patients may prevent ventilator-associated pneumonia. The goal of this study was to determine the minimum frequency (once or twice a day) for 0.12% chlorhexidine gluconate application necessary to reduce oral colonization by pathogens in 175 intubated patients in a trauma intensive care unit. METHODS A randomized, double-blind, placebo-controlled clinical trial tested oral topical 0.12% chlorhexidine gluconate or placebo (vehicle alone), applied once or twice a day by staff nurses. Quantitation of colonization of the oral cavity by respiratory pathogens (teeth/denture/buccal mucosa) was measured. RESULTS Subjects were recruited from 1 March, 2004 until 30 November, 2007. While 175 subjects were randomized, microbiologic baseline data was available for 146 subjects, with 115 subjects having full outcome assessment after at least 48 hours. Chlorhexidine reduced the number of Staphylococcus aureus, but not the total number of enterics, Pseudomonas or Acinetobacter in the dental plaque of test subjects. A non-significant reduction in pneumonia rate was noted in groups treated with chlorhexidine compared with the placebo group (OR = 0.54, 95% CI: 0.23 to 1.25, P = 0.15). No evidence for resistance to chlorhexidine was noted, and no adverse events were observed. No differences were noted in microbiologic or clinical outcomes between treatment arms. CONCLUSIONS While decontamination of the oral cavity with chlorhexidine did not reduce the total number of potential respiratory pathogens, it did reduce the number of S. aureus in dental plaque of trauma intensive care patients.