This study evaluated the antibacterial efficacy, biofilm inhibition capabilities, and synergistic potential of copper oxide nanoparticles (CuONPs) against biofilm-forming Klebsiella pneumoniae, uniquely exploring previously unexplored mechanisms, particularly their influence on critical biofilm-associated gene regulatory networks. CuONPs were characterized by Atomic Force Microscopy (AFM) and Field Emission Scanning Electron Microscopy (FE-SEM), with antibacterial activity assessed through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Synergistic effects with antibiotics (Clindamycin, Bactrim, and Imipenem) were evaluated using disc diffusion methods, while biofilm inhibition was quantified via microtiter plate assays and bacterial reduction rates were assessed by time-kill kinetics. Additionally, qRT-PCR was employed to analyze expression changes of biofilm-related genes (luxS, mrkA, fimA, rcsA, and kpa). Results demonstrated substantial antibacterial activity of CuONPs against K. pneumoniae with consistent MIC and MBC values at 50 mg/ml and 100 mg/ml, respectively, significant antibiotic synergism with reductions in required dosages by 40%-60%, notable biofilm impairment up to 80%, rapid bacterial decline within 2-4 hours, and significant downregulation of biofilm-associated genes mrkA, fimA, and luxS (up to 5-fold). In conclusion, CuONPs exhibit pronounced antibacterial and anti-biofilm properties, significantly enhanced by antibiotic synergy, providing novel insights into biofilm regulatory mechanisms and highlighting their clinical potential for treating multidrug-resistant K. pneumoniae infections.