Abstract: To investigate the antibacterial activity of Allicin E (ALE) against Vibrio parahaemolyticus, the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and growth curve were determined. The antibacterial mechanisms were investigated by exploring the influence of ALE on the extracellular β-D-galactosidase activity, protein content, bacterial morphological structure, biofilm formation ability, and transcriptome of V. parahaemolyticus. The results showed that the MIC and MBC of ALE against V. parahaemolyticus were 7.8 and 31.2 μg/mL, respectively. Treatment with ALE resulted in damage to the cell wall and membrane structure of V. parahaemolyticus, leading to the leakage of intracellular substances. Compared with the control group, extracellular β-D-galactosidase activity and protein content in the ALE-treated group were significantly increased (P<0.05), while the amount of bacterial biofilm formation was significantly decreased (P<0.05). The transcriptome analysis results showed that 145 genes were significantly downregulated in the ALE group, mainly enriched in pathways such as MAPK signal transduction, flagella assembly, and biofilm formation in Pseudomonas aeruginosa. Conversely, 133 genes were significantly upregulated, mainly related to amino acid metabolism and degradation pathways. The RNA-seq results were consistent with the RT-qPCR validation tests. Our results demonstrated that ALE showed a strong antimicrobial effect against V. parahaemolyticus by disrupting bacterial structure, inhibiting biofilm formation, and regulating the expression of genes such as flagella and biofilms. This study laid an important foundation for exploring the role and mechanism of ALE against V. parahaemolyticus.