Biofilm spoilage has become one of the most common concerns in fermented foods. The objective of the present study was to explore the biofilm-inhibitory effect and antibiofilm mechanism of dielectric barrier discharge (DBD) cold plasma on Pichia manshurica (a species of yeast responsible for biofilm spoilage in fermented foods). After plasma treatment (80 kV, 50 Hz) for 4.5 min and 7.5 min, viable counts decreased by 2.38 and 5.11 lg CFU/mL, respectively, and the biofilm-forming rate decreased to 73% and 48% of the control group, respectively, in microbiological media. In addition, DBD caused severe morphological damage, cell membrane permeabilization and metabolic changes. A number of amino acid metabolic pathways, the tricarboxylic acid cycle (TCA), and the synthesis of extracellular polymeric substance (EPS) were destroyed. The cell damage and changes in multiple metabolic activities led to significant inhibitory effects on Pichia manshurica and its biofilm-forming ability. This study found that DBD can significantly inhibit the biofilm-forming ability of Pichia manshurica (a species of yeast responsible for biofilm spoilage in fermented foods) by causing severe cell damage and metabolic changes. The exploration of the biofilm inhibitory effect of DBD on Pichia manshurica and its mechanism can provide a theoretical basis for the application of DBD technology in the inhibition of biofilm spoilage in foods. [Display omitted] • The viable counts of Pichia manshurica decreased by 2.38 and 5.11 lg CFU/mL after Dielectric barrier discharge (DBD) treatment for 4.5 and 7.5 min, respectively, using microbiological media. • The biofilm forming rate decreased to 73% and 48% of the control group after DBD treatment for 4.5 and 7.5 min, respectively, using microbiological media. • The treated yeasts exhibited severe morphological damage and cell membrane permeabilization. • DBD treatment interfered with the TCA cycle, several amino acid metabolic pathways and the synthesis of extracellular polymeric substance (EPS). [ABSTRACT FROM AUTHOR]