Background: Multidrug-resistant (MDR) Pseudomonas aeruginosa strains, particularly those forming robust biofilms, severely complicate urinary tract infections (UTIs). Green nanotechnology offers a sustainable solution to this crisis.

Methods: In this cross-sectional study (2025), 103 P. aeruginosa isolates were collected from UTI patients in Zahedan, Iran. Following phenotypic and molecular (16S rRNA) identification, biofilm formation was assessed via microtiter plate assay. Silver nanoparticles (AgNPs) and copper oxide nanoparticles (CuONPs) were biosynthesized using Mesembryanthemum nodiflorum extract and characterized by UV-Vis, TEM, and XRD. Antimicrobial and antibiofilm activities were evaluated against strong biofilm producers. Expression levels of lasI and pelA genes were quantified by Real-time PCR, and cytotoxicity was tested on HEK293 cells.

Results: Characterization confirmed stable, spherical AgNPs (~45 nm) and CuONPs (~15 nm). Despite the larger size of AgNPs, they exhibited superior efficacy with lower MIC values (6.25–12.5 µg/mL) and higher biofilm inhibition (73–75%) compared to smaller CuONPs (MIC 25–50 µg/mL; 55–60% inhibition). AgNPs significantly downregulated lasI (4.7-fold) and pelA (4.0-fold) genes, outperforming CuONPs. Both agents maintained >85% cell viability at therapeutic doses.

Conclusion: Although smaller nanoparticles are typically expected to be more reactive, this study revealed that AgNPs possessed markedly stronger antibiofilm and gene-suppressive activities than the smaller CuONPs. This suggests that intrinsic material properties override particle size in determining efficacy. These green-synthesized nanoparticles offer a promising, biocompatible strategy for coating urinary catheters to combat MDR infections.