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The inhibitory and bactericidal activities of thyme oil against the foodborne multiple antibiotics-resistant Enterococcus faecalis biofilm were evaluated in this study. Gas chromatography-mass spectrometry revealed that more than 70% of the composition of thyme oil is thymol. Crystal violet staining assay showed that 128 and 256 mg/mL thyme oil significantly inhibited the biofilm formation of E. faecalis. The cell adherence of E. faecalis, as shown by its swimming and swarming motilities, was reduced by thyme oil. The exopolysaccharide (EPS) quantification assay showed that thyme oil inhibited the EPS synthesis in E. faecalis biofilms. The 3D-view observations through confocal laser scanning and scanning electron microscopy suggested that cell adherence and biofilm thickness were decreased in thyme oil-treated biofilms. Quantitative real-time analyses showed that the transcription of ebp and epa gene clusters, which were related to cell mobility and EPS production, was inhibited by thyme oil. Thus, thyme oil effectively inhibited the biofilm formation of E. faecalis by affecting cell adherence and EPS synthesis. Furthermore, 2,048 and 4,096 mg/mL thyme oil can effectively inactivate E. faecalis population in the mature E. faecalis biofilms by 5.75 and 7.20 log CFU/mL, respectively, after 30 min of treatment. Thus, thyme oil at different concentrations can be used as an effective antibiofilm or germicidal agent to control E. faecalis biofilms. [ABSTRACT FROM AUTHOR]