Virulent strain Pseudomonas aeruginosa isolated from Mahananda River exhibited the highest hemolytic activity and virulence factors and was pathogenic to fish as clinical signs of hemorrhagic spots, loss of scales, and fin erosions were found. S3 was cytotoxic to the human liver cell line (WRL-68) in the trypan blue dye exclusion assay. Genotype characterization using whole genome analysis showed that S3 was similar to P. aeruginosa PAO1. The draft genome sequence had an estimated length of 62,69,783 bp, a GC content of 66.3%, and contained 5916 coding sequences. Eight genes across the genome were predicted to be related to hemolysin action. Antibiotic resistance genes such as class C and class D beta-lactamases, fosA, APH, and catB were detected, along with the strong presence of multiple efflux system genes. This study shows that river water is contaminated by pathogenic P. aeruginosa harboring an array of virulence and antibiotic resistance genes which warrants periodic monitoring to prevent disease outbreaks.