Multidrug-resistant (MDR) Escherichia coli is a major foodborne pathogen posing a critical threat to public health, particularly through the contamination of animal products. The increasing prevalence and virulence of MDR E. coli strains underscore the urgent need for alternative antimicrobial strategies. This study aimed to synthesize and characterize zinc oxide nanoparticles (ZnO-NPs) using Stevia rebaudiana as a sustainable capping and reducing agent, aligning with green chemistry principles. Of the 120 fish samples, 74.2% (89/120) were positive for E. coli contamination. Among the identified E. coli strains, 77.8% (119/153) were classified as MDR. Resistance profiling revealed 22 distinct patterns, and seven highly resistant and virulent strains were selected for further analyses. The eco-friendly auto-combustion synthesis of ZnO-NPs produced nanoparticles with semi-spherical to hexagonal shapes and an average size ranging from 12 to 25 nm. Scanning Electron Microscope–Energy Dispersive X-ray analysis (SEM-EDS) confirms that ZnO-NPs primarily consist of zinc (37.5%) and oxygen (19.9%), with carbon (42.6%) indicating the green synthesis process. ZnO-NPs demonstrated potent, dose-dependent antibacterial and antibiofilm activity against the selected MDR E. coli strains. Additionally, mechanistic studies revealed that ZnO-NPs disrupt bacterial cell membranes, alter cellular morphology, and interfere with DNA integrity. These findings highlight the potential of eco-friendly ZnO-NPs as a promising nanomaterial for enhancing food safety and addressing the growing challenge of MDR foodborne bacteria.

The rise of multidrug-resistant (MDR) bacteria in food products poses a significant threat to public health, necessitating innovative and sustainable antimicrobial solutions. This study investigates the green synthesis of zinc oxide nanoparticles (ZnO-NPs) using Stevia rebaudiana extracts to evaluate their antibacterial and antibiofilm activities against MDR Staphylococcus aureus strains isolated from sold fish samples. The obtained results show that the contamination with S. aureus reached 54.2% in the tested fish samples (n = 120), underscoring the urgent need for effective interventions. ZnO-NPs were successfully synthesized and characterized using UV-visible spectroscopy, FT-IR, XRD, and TEM, confirming their formation with an average size of 15.7 nm and reflecting their suitability for antimicrobial and biological applications. ZnO-NPs exhibited potent antibacterial activity, with a maximum inhibition zone of 24.4 ± 0.4 mm at 20 μg/disk, MIC values of 6.25–25 μg/mL, and MBC values of 12.5–50 μg/mL. Additionally, biofilm formation was inhibited by up to 92.1% at 250 μg/mL. Our mechanistic study confirmed that ZnO-NPs damage bacterial membranes and DNA, leading to the intracellular leakage of cell components that lead to bacterial cell lysis. The use of S. rebaudiana in ZnO-NP synthesis aligns with green chemistry principles, offering an eco-friendly alternative to conventional antibiotics and enhancing the bioactivity of ZnO-NPs, and may address the growing issue of antimicrobial resistance, thereby contributing to improved food safety and public health protection.