Abstract In cystic fibrosis (CF), infections with Pseudomonas aeruginosa or other typical pathogens play a critical role in eliciting disease progression, leading to tissue damage and finally loss of lung function. Previous observations showed that the presence of various commensal bacteria and a higher airway microbiome diversity were associated with better lung function and less severe disease burden. Thus, the hypothesis was raised that commensal bacteria might be able to interfere with pathogenic bacteria. In this study, we aimed to identify airway commensal bacteria that inhibit the growth of P. aeruginosa.Through a screening experiment of co-culture with P. aeruginosa PAO1, we could identify more than 30 CF commensal strains from various species that inhibited the growth of P. aeruginosa. With multiple selected strains, we further verified the results with P. aeruginosa CF isolates and several other pathogens isolated from CF patients, and most of the identified commensal strains showed consistent results strongly inhibiting the growth of diverse CF pathogens.The underlying mechanisms of the growth-inhibition effects were first investigated through genomic analysis by comparing strains with and without growth-inhibition effects, which revealed that genes responsible for carbohydrate transport and metabolism were highly enriched in the inhibitory commensals. Metabolite analysis and functional analysis showed that commensals with inhibitory effects produce large amounts of acetate. Exogenous addition of acetate under a low pH inhibited the growth of P. aeruginosa, indicating acetate produced and released by commensals may affect the growth of P. aeruginosa living in the same microenvironment.In summary, through co-culture of P. aeruginosa with commensals, we could identify that a variety of airway commensal strains can inhibit the growth of P. aeruginosa by producing acetate. The data provide insights into possible novel strategies for controlling infections in people with CF and also emphasize the importance of preserving airway commensals when designing infection treatment strategies.