Introduction: Pseudomonas aeruginosa (P. aeruginosa) is a multidrug-resistant opportunistic pathogen frequently encountered in clinical settings, posing substantial challenges for effective treatment. Its resistance often arises from mutations in critical drug targets, such as penicillin-binding protein-3 (PBP3), a key β-lactam target essential for bacterial survival. The F533L mutation is linked to resistance against multiple antibiotics, underscoring the need for alternative therapeutic strategies. Methods: This study employed molecular docking to analyze the interactions between zinc oxide (ZnO) and copper oxide (CuO) nanoparticles (NPs) and the mutated PBP3 (F533L) of P. aeruginosa. Additionally, computational approaches were used to assess the impact of the F533L mutation on the structural stability of PBP3. Binding energy and interaction analyses were conducted to evaluate the PBP3-nanoparticle complexes. Results: The F533L mutation was found to alter the stability of PBP3, potentially contributing to resistance mechanisms. Molecular docking revealed that ZnONPs and CuONPs demonstrated binding energies of -2.15 kcal/mol and -1.48 kcal/mol, respectively. Both nanoparticles formed hydrogen bonding interactions with the mutated residue 533L, highlighting their potential to mitigate resistance. Conclusions: The findings suggest that ZnO and CuO nanoparticles could serve as promising therapeutic agents against antibiotic resistance in P. aeruginosa, particularly targeting the F533L mutation in PBP3. Further experimental and in vitro studies are essential to validate their therapeutic efficacy and broader applications.