Abstract: To identify the primary spoilage molds in moldy whole wheat bread and to investigate the antimicrobial mechanism of citral, this study employed naturally spoiled whole wheat bread as the experimental material. The dominant spoilage fungi were isolated and identified through dilution plating, morphological observation, and rDNA-ITS sequence analysis. Subsequently, the ultrastructural alterations of fungal hyphae induced by citral were observed using scanning electron microscopy (SEM). Additionally, the disruption of cell membrane integrity was assessed by measuring nucleic acid leakage, extracellular conductivity, metal ion concentrations, and ergosterol content. The antimicrobial mechanism of citral was comprehensively analyzed by assessing chitinase activity, mitochondrial membrane potential, oxidative stress-related enzymes (superoxide dismutase (SOD), glutathione S-transferase (GST), catalase (CAT)), and malondialdehyde (MDA) levels. The results revealed that Penicillium expansum and Aspergillus niger were the predominant spoilage molds in whole wheat bread. Citral exhibited dose-dependent inhibitory effects, with minimum inhibitory concentrations (MIC) of 0.40 μL/mL for P. expansum and 0.35 μL/mL for A. niger. SEM analysis demonstrated that MIC-level citral treatment induced severe shrinkage, distortion, and fragmentation in P. expansum hyphae, while A. niger hyphae exhibited surface collapse and pore formation. Mechanistic studies indicated that citral: (1) disrupted cell wall integrity by enhancing chitinase activity (5.37- and 4.23-fold increases for P. expansum and A. niger, respectively); (2) compromised membrane stability via suppressing ergosterol biosynthesis (50.01%-51.39% reduction); (3) induced oxidative stress by significantly increasing SOD and GST activities while inhibiting CAT activity, leading to reactive oxygen species accumulation and lipid peroxidation (67.83%-153.33% increase in MDA); and (4) impaired mitochondrial function through depolarization of membrane potential. These synergistic effects altered membrane permeability, triggering the leakage of Na⁺ and K⁺ ions as well as intracellular nucleic acids. Collectively, citral exerted antifungal activity by damaging the cell wall/membrane system and disrupting intracellular redox homeostasis. This study elucidates the molecular mechanism of citral’s antifungal action, providing a theoretical foundation for developing plant-derived preservatives for cereal-based food products.