Antibiotic resistance is one of the most pressing threats to human health, yet recent work highlights how loss of resistance may also drive pathogenesis in some bacteria. In two recent studies, we found that β-lactam antibiotic and nutrient stresses faced during infection selected for the genetic inactivation of the () antibiotic efflux pump . Unexpectedly, efflux pump mutations increased virulence during infection; however, neither the prevalence of efflux pump inactivating mutations in real human infections, nor the mechanisms driving increased virulence of efflux pump mutants are known. We hypothesized that human infection would select for efflux pump mutations that drive increased virulence in clinical isolates. Using genome sequencing of hundreds of clinical isolates, we show that efflux pump inactivating mutations are enriched in cystic fibrosis isolates relative to intensive care unit clinical isolates. Combining RNA-seq, metabolomics, genetic approaches, and infection models we show that efflux pump mutants have elevated expression of two key virulence factors, elastase and rhamnolipids, which increased virulence and lung damage during both acute and chronic infections. Increased virulence factor production was driven by higher Pseudomonas quinolone signal levels in the efflux pump mutants. Finally, genetic restoration of the efflux pump in a representative ICU clinical isolate and the notorious CF Liverpool epidemic strain reduced their virulence. Together, our findings suggest that mutations inactivating antibiotic resistance mechanisms could lead to greater patient mortality and morbidity.