We studied the influences of antimicrobial agents on the colonization of the respiratory tract and infection with Enterococcus faecalis in intensive care unit (ICU) patients receiving mechanical respiration for at least 3 days. In a matched-cohort analysis, patients receiving topical antimicrobial prophylaxis (TAP) of the oropharynx and stomach with antimicrobial agents not treating E. faecalis were compared with patients not receiving TAP. Patients were matched with controls on the basis of their duration in the ICU, their use of systemic antibiotics treating and not treating E. faecalis, the administration of TAP, their APACHE II score, and surgical procedures they had undergone. In all, 276 patients were analyzed. The colonization of the oropharynx and/or trachea by E. faecalis at admission was demonstrated for 43 patients (16%). Twenty patients (9%) acquired tracheal colonization and 91 patients (40%) acquired oropharyngeal colonization with E. faecalis. In the matched-cohort analysis, 43 patients receiving TAP were matched in two controls each. TAP patients more frequently acquired tracheal colonization (15 of 43 versus 2 of 86 patients, P < 0.00001) and infections with E. faecalis (6 of 43 versus 1 of 86 patients, P < 0.01). The use of topical antibiotics and treating E. faecalis increased the risk for colonization and infection with E. faecalis.