Background: Biofilm formation is a major challenge in healthcare, as bacteria encased in a protective matrix become highly resistant to antibiotics and immune defenses. Which urgently required a highly attention Objectives: This study investigates the antibiofilm activity of zinc oxide nanoparticles (ZnO NPs) and low frequency sonication against multidrug- resistant (MDR) bacterial isolates. Material and methods: Bacterial isolates were identified, including Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus, which were isolated form different clinical samples. E. coli strains were nominated to synthesize the ZnO NPs. Production of the Zn nanoparticles were confirmed using various professional analysis applications using UV-Visible spectroscopy, FTIR, XRD, and SEM. Results: treatment using 40 kHz ultrasound bath alone showed a 20% reduction in the ability of the K.pneumoniae of biofilm production after 30 min. ZnO nanoparticles (ZnO NPs) at MIC (12.5 mg/ml) exhibit clear antibiofilm activity 48.68% reduction, while the subMIC concentration revealed only 20.3%., but their efficacy is significantly enhanced when combined with 40 kHz ultrasound, leading to up to 18% biofilm inhibition against Klebsiella pneumoniae after 24 hours. Notably, sub-MIC below (12.5mg/ ml) of ZnO NPs exhibit a fluctuating effect on gene expression, for several biofilm-associated genes (FimH, K2A( as analyzed by real-time PCR (RT-PCR). Conclusion: These findings underscore the potential of combined therapy of ultrasound and ZnO NPs as a cost-effective, eco-friendly alternative for managing (MDR) bacteria and biofilm-related diseases, with implications for future therapeutic applications.