Aim The aim of this study was to investigate the natural variation in the antibiotic sensitivity, biofilm formation and virulence among Pseudomonas aeruginosa isolated from the catheter-associated urinary tract infection (CAUTI) from a single centre. Methods and Results Pseudomonas aeruginosa strains were isolated from the patients with CAUTI after obtaining informed consent. These isolates were identified by routine biochemical methods and 16S rRNA gene sequencing. Antibiotic sensitivity and virulence factors were measured using standard protocols. Crystal violet staining, scanning electron microscopy and confocal laser scanning microscopy were used for the biofilm studies. The extent of infectivity of the strains to induce cell lysis was studied in vitro using the Human Embryonic Kidney cells (HEK 293T). Association between virulence factors, biofilm formation and antibiotic resistance among the strains was analysed statistically. Among the 1266 patients admitted during the 2016-2017 period, 98 cases of CAUTI were reported and 18 center dot 36% (n = 18) was due to P. aeruginosa infection. Antibiogram showed that 94 center dot 4% of isolates were resistant to multiple antibiotics and 73 center dot 7% were carbapenem-resistant. All the isolates formed biofilm on different material surfaces with varying intensity (OD580 >= 0 center dot 20-1 center dot 11). The biofilm intensity on silicone-latex material was significantly higher compared to the polystyrene surface (P > 0 center dot 05). All the strains were highly virulent and able to cause cell killing of HEK 293T cells with a rate ranging from 69 center dot 35 to 100% and showed very low sensitivity to healthy human serum. Conclusions Antibiotic sensitivity and association between the virulence factors and biofilm formation in the P. aeruginosa clinical strains showed complex natural diversity. Significance and Impact of the Study This study demonstrates the natural diversity and adaptation in virulence factors, biofilm formation and host-pathogen interaction among catheter-associated P. aeruginosa strains. The findings from the study urge for developing individualized drug strategy for targeting these multidrug-resistant pathogens.