Respiratory syncytial virus (RSV) is the major pathogen responsible for acute bronchiolitis in infancy. However, evaluation of the relative importance of rhinovirus or multiple viral infections has been hampered by the lack of sensitive diagnostic methodologies. Therefore, in this study we used the reverse transcription-polymerase chain reaction for 11 respiratory pathogens to assess the etiology in infants with acute bronchiolitis and correlate it with clinical characteristics of the disease. Viruses were detected in 73.7% of patients. RSV was identified in 72.4% of virologically confirmed cases, rhinovirus in 29%, whereas multiple infections represented 19.5% of cases, most of which (69%) were combinations of rhinovirus with RSV. In a logistic regression model controlling for age, sex, birth weight, presence of fever, and day of disease on admission, the presence of rhinovirus was found to increase by approximately five-fold, the risk for severe disease. Multiple pathogens had a similar trend in the univariate analysis, which was eliminated in the multivariate model. Multiple virus cases were admitted to the hospital later in the course of their disease than unique pathogen cases, suggesting successive infections. In conclusion, rhinovirus is second only to RSV as a causative agent of bronchiolitis and is associated with more severe disease. The presence of more than one pathogen may influence the natural history of acute bronchiolitis.