The increasing prevalence of antimicrobial resistance requires a continuous effort to find new antimicrobials. Whole-genome, single-gene knockout libraries such as the University of Washington two-allele library of Pseudomonas aeruginosa seem well suited for identifying new antimicrobial targets. However, we recently noticed with the P. aeruginosa two-allele mutant library that intrinsic, unidentified factors may affect susceptibility for some antimicrobials with many randomly picked mutants, thereby obscuring effects attributed to specific candidate mutants. For instance, a phoU mutant conferred hypersensitivity to quinolones as expected, but 10 unrelated mutants also exhibited this phenotype, and PCR failed to confirm transposition inside the phoU allele. Colony color analysis indicated more phenazine pigments in mutants, yet pyocyanin-nonproducing mutants still had reduced MICs. Transposon-mediated polarity was an unlikely explanation. Hypersusceptibility was observed with chloramphenicol but not with tobramycin, meropenem, or ceftazidime. The results suggest transposition/insertion of a DNA fragment into the P. aeruginosa chromosome may itself have a general effect on susceptibility to some antimicrobials. Thus, interpretation of antimicrobial experiments involving mutants from the P. aeruginosa insertional libraries requires caution.