The increasing prevalence of drug-resistant bacteria is attributed to the extensive use of antibiotics, which causes selective pressure on the nasopharyngeal bacterial flora. Shortened courses of antibiotics have been proposed to decrease the development of resistant strains. We determined the effect of a single intramuscular dose of ceftriaxone (50 mg/kg) on the nasopharyngeal bacterial flora in 167 children (median age 13 mo) with acute otitis media. Nasopharyngeal samples for bacterial culture were obtained before and 5 d after treatment with ceftriaxone. Before treatment, Moraxella catarrhalis was isolated in 99 (59%) children, Streptococcus pneumoniae in 87 (52%), and Haemophilus influenzae in 53 (32%). After treatment, M. catarrhalis was found in 62 (37%) children, which constitutes a 37% decrease in the colonization rate by this pathogen (p < 0.001). S. pneumoniae was isolated in 50 (30%; 43% decrease) and H. influenzae in 17 (10%; 68% decrease) children after treatment (p < 0.001 for both). Before treatment, 60% of pneumococcal isolates were sensitive to penicillin, 26% were of intermediate susceptibility, and 14% were penicillin-resistant. Eradication of S. pneumoniae occurred mainly in children with penicillin-sensitive isolates. As a consequence, only 24% of pneumococcal isolates that remained after treatment were sensitive to penicillin, 59% were penicillin-intermediate, and 16% were penicillin-resistant. A single dose of ceftriaxone resulted in significant changes in the nasopharyngeal bacterial flora, increasing the relative prevalence of pneumococcal strains with decreased susceptibility to penicillin.