The influence of topical antimicrobial prophylaxis (TAP) on colonization of oropharynx and trachea was studied in patients receiving and not receiving prophylaxis. Twenty-two patients in Intensive Care Unit (ICU) I (Group 1) received TAP (tobramycin, colistin, and amphotericine B in oropharynx and stomach). Simultaneous to Group 1, 21 patients (Group 2) not receiving TAP were studied in ICU I. A control group of patients admitted to another, identical, ICU (ICU II), where no TAP was administered, were studied simultaneously (Group 3a, n = 23). A second control group (Group 3b, n = 31), was formed by collecting data from patients admitted to ICU I in Period II. Patients receiving TAP were less frequently colonized than patients not receiving prophylaxis. Moreover, of the patients not receiving TAP, those staying in the ICU where TAP was administered (Group 2) were less frequently colonized than patients in another ICU (Group 3). Of the patients not colonized on admission, those staying in the ICU where TAP was administered remained free of colonization for a longer time. In the ICU where no TAP was administered, more patients were colonized simultaneously and cross-acquisition occurred more frequently. TAP significantly influenced colonization of oropharynx and trachea in patients receiving and not receiving prophylaxis within the same ICU as compared with patients not receiving prophylaxis in another identical ICU.