Infectious keratitis continues to be a prominent cause of vision impairment worldwide through a variety of causes. Pseudomonas aeruginosa is a Gram-negative bacterium that frequently causes vision-threatening microbial keratitis. P. aeruginosa contains a diverse array of virulence factors, including exoA, exoS, nan1, and lasB, some of which may contribute to its pathogenicity. Because the clinical characteristics of bacterial keratitis vary, making a quick differential diagnosis can be difficult, resulting in a delay in diagnosis and worse outcome. In this study, we performed multiplex polymerase chain reaction to detect the presence of nan1, toxA, exoS, and lasB, and determine their association with distinct clinical presentations of P. aeruginosa-related keratitis. We also performed antibiotic susceptibility testing of the isolates. A total of 49 P. aeruginosa strains were obtained from individuals with keratitis between May 2021 and December 2021 from the Research Institute of Ophthalmology, Giza, Egypt. Results showed that lasB was most expressed gene (81.8%), followed by tox (63.6%) and exoS (31.8%), whereas nan1 was the least commonly expressed gene 1316 (22.7%). The antibiotic susceptibility profile showed that TOB was the least sensitive antibiotic (26.5%), followed by CIP (34.7%), whereas CT was the most sensitive antibiotic (89.8%), followed by GAT (83.7%) and PB (81.6%). Several virulence genes were identified in P. aeruginosa isolates, suggesting that these genes are associated with varying degrees of intrinsic virulence and pathogenicity. Substantial associations between specific virulence genes and the source of infection imply that infection control measures can aid in regulating the distribution of virulence genes among P. aeruginosa strains.