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Background: Streptococcus mutans is a major component of dental plaque, contributing to cariogenic biofilm formation and inducing dental caries. Attempts have recently been made to use postbiotic mediators (PMs) to prevent dental caries. This research evaluated the antimicrobial/antibiofilm activity of PMs derived from Lactobacillus rhamnosus GG (LGG) and Lactobacillus reuteri (LR) against S. mutans in vitro. Methods: PMs were obtained from the Lactobacilli supernatants. The minimum inhibitory concentration, minimum bactericidal concentration, antibiofilm potential, and metabolic activity of PMs against S. mutans were evaluated using CFU/mL, scanning electron microscopy, and XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction assay. The expression of gtfB gene as one of the most important genes involved in S. mutans biofilm formation was also measured using qRT-PCR. Results: CFU score was reduced by both PMs, but the reduction was only significant in LGG (p = 0.02). Both PMs caused a significant decrease in the metabolic activity of S. mutans compared with the controls (p ≤ 0.002). S. mutans treated with LGG PMs exhibited more destructive effects than LR PMs (p > 0.05). S. mutans gtfB gene expression was significantly downregulated when treated with the PMs obtained from both LGG and LR (p = 0.01 for both). Conclusions: We showed that PMs isolated from two Lactobacillus strains inhibited S. mutans biofilm, metabolic activity, and gtfB gene expression. Therefore, these derivatives may be a suitable biofilm-destruction agent against S. mutants. However, the oral environment is a complex ecosystem that needs further investigation.