Abstract BACKGROUND Walnut meal is a by‐product of walnut oil extraction. Its fermented product exhibits antibacterial activity against Staphylococcus aureus , mainly due to the produced peptides during fermentation. This study aimed to identify antibacterial peptides from fermented walnut meal (FW) targeting S. aureus , focusing on cell membrane and energy metabolism effects. RESULTS FW reduced the aerobic plate count of tomato juice by 75.45%. Peptides with a net positive charge and mean hydrophobicity greater than 0 accounted for 63.7% and 35.92%, respectively. Molecular docking identified three antibacterial peptides – HAAPK (HK‐5), TYFPH (TH‐5), and WVSK (WK‐4) – that bind penicillin‐binding proteins and critical membrane protein amino acids via hydrogen bonds, hydrophobic interactions, and π–π conjugate interactions. HK‐5 exhibited the highest antibacterial activity. Structural analyses using Fourier transform infrared and circular dichroism spectroscopy revealed that HK‐5 predominantly adopts β‐sheets (45.0%) in solid state and random coils (51.2%) in aqueous environments. Treatment with 1.0 minimum inhibition concentration of HK‐5 reduced intracellular protein, pyruvate, and β‐galactosidase activity by 55.0%, 76.84%, and 68.57%, respectively, while lipase activity increased by 18.25%, indicating membrane damage. Additionally, adenosine triphosphate content and the activities of succinate dehydrogenase and malate dehydrogenase decreased by 93.28%, 82.63%, and 86.89%, respectively, whereas isocitrate dehydrogenase activity leakage nearly doubled, pointing to energy metabolism disturbances. Scanning and transmission electron microscopy confirmed morphological and ultrastructural damage caused by HK‐5. CONCLUSION These findings highlight FW as a promising source of antibacterial agents, providing a sustainable use for agricultural waste. © 2025 Society of Chemical Industry.