Endogenous transporters protect against antibiotics and also contribute to bacterial defense from environmental toxins. We evaluated the effect of overexpression of four efflux pumps, NorA, NorB, NorC, and Tet38, on survival following exposure to pyocyanin (PYO) of , using a well diffusion assay. We measured the PYO-created inhibition zone and found that only an overexpression of NorA reduced susceptibility to pyocyanin killing. The MIC of the NorA overexpressor increased threefold compared to that of wild-type RN6390 and was reduced 2.5-fold with reserpine, suggesting that increased NorA efflux caused PYO resistance. The PYO-created inhibition zone of a mutant was consistently larger than that of a plasmid-borne NorA overexpressor. PYO also produced a modest increase in expression (1.8-fold at 0.25 µg/mL PYO) that gradually decreased with increasing PYO concentrations. Well diffusion assays carried out using showed that mutant was less susceptible to killing by PYO-deficient mutants PA14 and PA14 than to killing by PA14. NorA overexpression led to reduced killing by all tested . We evaluated the NorA-PYO interaction using a collection of 22 clinical isolates from adult and pediatric cystic fibrosis (CF) patients, which included both (CF-SA) and (CF-PA). We found that when isolated alone, CF-PA and CF-SA expressed varying levels of PYO and transcripts, but all four CF-PA/CF-SA pairs isolated concurrently from CF patients produced a low level of PYO and low transcript levels, respectively, suggesting a partial adaptation of the two bacteria in circumstances of persistent co-colonization.