Cystic fibrosis (CF) results in accumulation of mucus in the airways that promotes pathogen colonization, leading to respiratory exacerbations, lung failure, and death. The CF airway harbors a complex microbiota, including pathogens and oropharyngeal bacteria. Here, we reanalyzed 5,260 16S rRNA gene microbiota data sets to infer associations between members of the CF microbiota. We determined that pathogens are more likely to proliferate and dominate when present, while oropharyngeal bacteria are generally present at lower abundances. We found that higher diversity and increasing numbers of pairwise associations between bacteria were positively correlated with lung function. In contrast, dominance of the microbiota by pathogens was associated with loss of lung function. We determined that pathogens are largely negatively associated with each other and oropharyngeal bacteria, suggesting they are antagonistic toward other bacteria. As a first test of this hypothesis, we cultured 1,597 isolates from 96 people with CF and performed 12,542 coculture assays against eight representative pathogens and oropharyngeal bacteria, finding 23% of interactions resulted in inhibition. Among our isolates, Pseudomonas were the most inhibitory, but we observed variable activity between different isolates. We further found Pseudomonas aeruginosa isolates, even those from the same donor and time point, exhibited significant differences in their metabolome and bioactivity profiles that correlated with acquisition of mutations. Together, our results suggest that pathogens may disrupt the CF microbiota and proliferate in part through bioactive metabolite production. Furthermore, these data highlight that characterizing multiple isolates is necessary to capture the full landscape of chemically mediated interactions within microbiomes.