Virulent strains of Pseudomonas aeruginosa are often associated with an acquired cytotoxic protein, exoenzyme U (ExoU) that rapidly destroys the cell membranes of host cells by its phospholipase activity. Strains possessing the exoU gene are predominant in eye infections and are more resistant to antibiotics. Thus, it is essential to understand treatment options for these strains. Here, we have investigated the resistance profiles and genes associated with resistance for fluoroquinolone and beta-lactams. A total of 22 strains of P. aeruginosa from anterior eye infections, microbial keratitis (MK), and the lungs of cystic fibrosis (CF) patients were used. Based on whole genome sequencing, the prevalence of the exoU gene was 61.5% in MK isolates whereas none of the CF isolates possessed this gene. Overall, higher antibiotic resistance was observed in the isolates possessing exoU. Of the exoU strains, all except one were resistant to fluoroquinolones, 100% were resistant to beta-lactams. 75% had mutations in quinolone resistance determining regions (T81I gyrA and/or S87L parC) which correlated with fluoroquinolone resistance. In addition, exoU strains had mutations at K76Q, A110T, and V126E in ampC, Q155I and V356I in ampR and E114A, G283E, and M288R in mexR genes that are associated with higher beta-lactamase and efflux pump activities. In contrast, such mutations were not observed in the strains lacking exoU. The expression of the ampC gene increased by up to nine-fold in all eight exoU strains and the ampR was upregulated in seven exoU strains compared to PAO1. The expression of mexR gene was 1.4 to 3.6 fold lower in 75% of exoU strains. This study highlights the association between virulence traits and antibiotic resistance in pathogenic P. aeruginosa.