Biofilm represents an organized structure of microorganisms within an extracellular matrix attached to a surface. While the importance of biofilm in prosthetic heart valve and catheter-related infections has been known since the 1980s, the role of mucosal biofilm in human disease pathogenesis has only recently been elucidated. It is now clear that mucosal biofilm is present in both healthy and pathologic states. The purpose of this review is to examine the role of mucosal biofilm in pediatric respiratory infections. Mucosal biofilm has been implicated in relationship to several pediatric respiratory infections, including tonsillitis, adenoiditis, otitis media with effusion, chronic rhinosinusitis, persistent endobronchial infection, and bronchiectasis. In these conditions, core pathogens are detected in the biofilm, biofilm organisms are often detected by molecular techniques when conventional cultures are negative, and biofilm presence is more extensive in relation to disease than in healthy tissues. In chronic rhinosinusitis, the presence of polymicrobial biofilm is also a predictor of poorer outcome following sinus surgery. Biofilm in the tonsillar and adenoidal compartments plays a distinct role in contributing to disease in the middle ear and sinuses. Key observations regarding the relevance of biofilm to pediatric respiratory infections include (1) the association between the presence of biofilm and persistent/recurrent and more severe disease in these tissues despite antibiotic treatment, (2) linkage between biofilm core pathogens and acute infections, and (3) interrelationship between biofilm presence in one tissue and persistent or recurrent infection in an adjacent tissue. A greater understanding of the significance of mucosal biofilm will undoubtedly emerge with the development of effective means of eradicating mucosal biofilm.