The rise of multidrug-resistant (MDR) bacteria in food products poses a sig nificant 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 Received: 9 January 2025 Revised: 18 February 2025 Accepted: 25 February 2025 Published: 27 February 2025 Citation: Elabbasy, M.T.; El Bayomi, R.M.; Abdelkarim, E.A.; Hafez, A.E.-S.E.; Othman, M.S.; Ghoniem, M.E.; Samak, M.A.; Alshammari, M.H.; Almarshadi, F.A.; Elsamahy, T.; et al. Harnessing Stevia rebaudiana for Zinc Oxide Nanoparticle Green Synthesis: A Sustainable Solution to Combat Multidrug-Resistant Bacterial Pathogens. Nanomaterials 2025, 15, 369. https://doi.org/10.3390/ nano15050369 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). 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 success fully 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 suitabil ity 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 is sue of antimicrobial resistance, thereby contributing to improved food safety and public health protection.