Background: People with cystic fibrosis (CF) experience recurrent lung infections leading to frequent exacerbations and eventual respiratory failure. These exacerbations are associated with shifts in bacterial community, where certain species become dominant driving inflammation. In stable states, such dominance is uncommon, suggesting that microbial interactions play a key role in maintaining balance. Here, we examined CF sputum samples to explore potential microbe–microbe interactions that could influence community composition. Methods Two CF sputum samples were collected from patients one at the onset of pulmonary exacerbation and a second during a follow-up appointment. Isolates (approximately: n=66,000) were screened for competitive behaviour using overlay and cross-streak assays, as well as high-throughput competition screening of high-density microbial arrays with the Singer ROTOR+ platform, to identify interactions across multiple taxa. Results: Several Staphylococcus warneri and Staphylococcus pasteuri isolates demonstrated active inhibition of Staphylococcus aureus growth. Additionally, Streptococcus anginosus group isolates exhibited time-dependent suppression of S. aureus in co-culture assays. These findings reveal previously unrecognised interspecies antagonism within the CF lung microbiome, indicating that certain resident microbes can limit the growth of dominant pathogens. Conclusion: This study provides evidence of interspecies inhibition among CF airway isolates, highlighting that microbial competition may act as a regulatory force preventing pathogen overgrowth. Understanding these microbial dynamics could inform microbiome-based therapeutic approaches to mitigate infection and exacerbation in CF.