ABSTRACTCystic fibrosis (CF) is a human genetic disorder which results in a lung environment that is highly conducive to chronic microbial infection. Over the past decade, deep-sequencing studies have demonstrated that the CF lung can harbor a highly diverse polymicrobial community. We expanded our existingin vitromodel ofPseudomonas aeruginosabiofilm formation on CF-derived airway cells to include this broader set of CF airway colonizers to investigate their contributions to CF lung disease, particularly as they relate to the antibiotic response of the population. Using this system, we identified an interspecies interaction betweenP. aeruginosa, a bacterium associated with declining lung function and worsening disease, andStreptococcus constellatus, a bacterium correlated with the onset of pulmonary exacerbations in CF patients. The growth rate and cytotoxicity ofS. constellatus7155 andP. aeruginosaPA14 were unchanged when grown together as mixed biofilms in the absence of antibiotics. However, the addition of tobramycin, the frontline maintenance therapy antibiotic for individuals with CF, to a mixed biofilm ofS. constellatus7155 andP. aeruginosaPA14 resulted in enhancedS. constellatusbiofilm formation. Through a candidate genetic approach, we showed thatP. aeruginosarhamnolipids were reduced upon tobramycin exposure, allowing forS. constellatus7155 biofilm enhancement, and monorhamnolipids were sufficient to reduceS. constellatus7155 biofilm viability in the absence of tobramycin. While the findings presented here are specific to a biofilm ofS. constellatus7155 andP. aeruginosaPA14, they highlight the potential of polymicrobial interactions to impact antibiotic tolerance in unanticipated ways.IMPORTANCEDeep-sequencing studies have demonstrated that the CF lung can harbor a diverse polymicrobial community. By recapitulating the polymicrobial communities observed in the CF lung and identifying mechanisms of interspecies interactions, we have the potential to select the best therapy for a given bacterial community and reveal potential opportunities for novel therapeutic interventions. Using anin vitromodel of bacterial infection on CF airway cells, we tested how a particular polymicrobial community grows, damages human cells, and responds to antibiotics in single and mixed infections. We describe here the mechanism of an interspecies interaction between two pathogens in the CF lung,P. aeruginosaandS. constellatus, which is potentiated by a commonly prescribed antibiotic, tobramycin.