Abstract:[Background] Lactobacillus plantarum is a kind of important probiotic. Our previous study suggested that L. plantarum CCFM8724 could inhibit the double species biofilm of Streptococcus mutans and Candida albicans. However, the specific contributing substances remain unclear. [Objective] To evaluate the characteristics of the antibacterial components and explore the material basis of L. plantarum CCFM8724. [Methods] The influence of temperature, pH and other factors on metabolites were measured. Gas chromatography-mass spectrometry (GC-MS) was performed to analyze the composition of L. plantarum metabolites. Organic solvent extraction and ultrafiltration were further applied to initially separate and purify the fermentation supernatant. The metabolites inhibiting the double species biofilms were then identified by liquid chromatography-mass spectrometry (LC-MS). [Results] Through multivariate statistical analysis, the main difference metabolites of L. plantarum fermentation supernatant were organic acids (such as phenyllactic acid, acetic acid, hydroxyhexanoic acid and glyceric acid, etc.). After preliminary extraction, identification and function verification, the active ingredients were mainly organic acids and cyclic peptide compounds. [Conclusion] The supernatant of L. plantarum CCFM8724 can inhibit the mixed-biofilm through the synergistic effect of organic acids and cyclic peptides. This study provides a theoretical basis for the further production and application of L. plantarum effective components.