Shewanella putrefaciens is Gram-negative bacterium and important spoilage organism in aquatic products, negatively impacting the organoleptic properties of aquatic products. S. putrefaciens could form biofilm, which increases persistence and contamination in food system. Efficient antibiofilm strategies are urgently needed to reduce its presence in food environment. This study aimed to explore the impact of perillaldehyde on S. putrefaciens biofilm and the underlying mechanisms using transcriptomic analysis. Perillaldehyde remarkably reduced extracellular polymeric substance contents, inhibited metabolic activity of biofilm cells, disrupted bacterial motility, loose biofilm structure and decreased biofilm formation in food juice and on various surfaces (stainless steel, silicone, glass, razon clam and shrimp). Transcriptome analysis revealed that 553 differentially expressed genes were identified, among which 254 were down-regulated and 299 were up-regulated. The differentially expressed genes included ATP-binding cassette transporters, ribosome, two-component systems, resistance/nodulation/division efflux systems, quorum sensing, amino acid metabolism, biosynthesis and degradation pathways. The findings demonstrate antibiofilm properties of perillaldehyde against S. putrefaciens and indicate that perillaldehyde could be developed as an antibiofilm agent to mitigate existence and contamination of S. putrefaciens and to reduce associated food loss caused by this spoilage bacteria.