Dental plaque, a complex biofilm system established by cariogenic bacteria such as Streptococcus mutans (S. mutans), is the initiator of dental caries. Studies have found that the cell-free supernatant (CFS) of Lactobacilli could inhibit S. mutans biofilm formation. However, the main antibiofilm substance of the Lactobacilli CFS that acts against S. mutans is unclear. The present study found that the CFS of Lactobacillus plantarum (L. plantarum) ATCC 14917 had the strongest antibiofilm effect among the five tested oral Lactobacilli. Further bioassay-guided isolation was performed to identify the main antibiofilm substance. The antibiofilm effect of the end product, named 1-1-4-3, was observed and the structure of it was elucidated by using Q-TOF MS, 2D NMR and HPLC. The results showed that several components in the CFS had an antibiofilm effect; however, the effect of 1-1-4-3 was the strongest, as it could reduce the generation of exopolysaccharides and make the biofilm looser and thinner. After structure elucidation and validation, 1-1-4-3 was identified as a mixture of lactic acid (LA) and valine. Additionally, LA was shown to be the main antibiofilm substance in 1-1-4-3. In summary, this study found that the antibiofilm effect of the L. plantarum CFS against S. mutans was attributable to the comprehensive effect of multiple components, among which LA played a dominant role.