<b><i>Introduction:</i></b> Although current guidelines recommend against routine antibiotic prescription for acute exacerbation of bronchial asthma, children with acute exacerbation of asthma receive antibiotic treatment more frequently. In addition, those antibiotics are often prescribed only for exacerbation of asthma without concurrent bacterial infection. <b><i>Objective:</i></b> To clarify the association between antibiotic treatment and bacterial colonization in acute exacerbation of asthma, we investigated whether or not antibiotics affect the clinical condition, laboratory findings, and pharyngeal bacterial colonization in those patients. <b><i>Methods:</i></b> Potential bacterial pathogens were investigated in pharyngeal samples of 111 children with acute exacerbation of asthma (mean/median age: 2.8/2.6 years old, respectively). We collected clinical data, such as the duration of wheezing and antibiotic use, and measured the peripheral white blood cell counts, C-reactive protein, and serum levels of total and allergen-specific IgE. <b><i>Results:</i></b> Antibiotics were used in 50.5% patients with acute asthma exacerbation and included cephalosporin, penicillin, macrolide, and others. Episodes of wheezing were significantly longer in patients with antibiotic treatment than in those without it (6.7 ± 3.6 days vs. 6.0 ± 3.1, <i>p</i> = 0.044). Similarly, episodes of wheezing were significantly longer in moderate exacerbation patients with antibiotics than in those without them. Furthermore, in patients with <i>Streptococcus pneumoniae</i>, antibiotic treatment was associated with an extended duration of wheezing in cases of acute moderate exacerbation (7.0 ± 2.4 days vs. 4.8 ± 4.1, <i>p</i> = 0.043). <b><i>Conclusions:</i></b> These results suggest that antibiotic treatment in acute exacerbation of asthma might lead to longer asthmatic symptoms, specifically in patients with pharyngeal <i>S. pneumoniae</i> colonization.