Multidrug-resistant Pseudomonas aeruginosa poses a significant public health challenge, particularly in healthcare settings, owing to its ability to evade multiple antibiotics. This study focused on the molecular profile of P. aeruginosa isolates to identify their resistance mechanisms and explore potential therapeutic targets. To characterize P. aeruginosa, its molecular diversity, multidrug resistance, genetic factors, and drug targets for new therapies. In total of 20 clinical samples were obtained, including P. aeruginosa (n=14), from three healthcare centers in Lahore, Pakistan. Samples underwent MDR assessment via Kirby-Bauer disc diffusion. gyrA/parC genes were detected by PCR, sequenced. Natural compounds docked to study binding. Following 14 identifies P. aeruginosa isolates, 28 % (n=4) isolates (NF12, NF22, NF24, and NF84) showed MDR characteristics to fluoroquinolones, carbapenems, and colistin, confirmation by 16S rRNA gene sequencing. PCR and sequencing identified gyrA and parC genes conferring fluoroquinolone resistance. Phylogenetic analysis revealed close clustering with P. aeruginosa. Berberine Hydroxide strongly bound GyrA and ParC (–6.6, –6.8 kcal/mol), while Curcumin had moderate binding, inhibiting resistant strains. This study highlights multidrug-resistant P. aeruginosa and shows strong binding potential with berberine hydroxide. Future research should develop effective therapies to address clinical antibiotic resistance.