Dental caries is a multifactorial chronic infectious disease that impacts healthcare costs globally, caused by alterations of the plaque microbiome and proliferation of cariogenic Streptococcus mutans. Treatments targeting S. mutans, such as alternative strategies using probiotics, might be effective in preventing the development of dental caries. In this study, the probiotic formulation of Lactobacillus reuteri SGL01, vitamin C, and acerola was tested against S. mutans DSM20523. Antimicrobial activity was assessed by deferred antagonism and spot-on-lawn assays for L. reuteri SGL01. MIC and MBC of L. reuteri SGL01 cell-free supernatant (CFS), vitamin C, and acerola were determined with the microdilution method. Time–kill assays determined the bactericidal kinetics for each compound. The checkerboard method was used to evaluate the potential synergistic activity of CFS–vitamin C or CFS–acerola at scalar dilutions from 1 to 8X MIC. Lastly, antibiofilm activity was tested for each compound. Antimicrobial activity of L. reuteri SGL01 was first assessed by classic methods. MIC and MBC values differed for one dilution for all compounds, with values of 25% and 50% for CFS, 9.3 mg/mL and 18.7 mg/mL for vitamin C, and 18.7 mg/mL and 37.5 mg/mL for acerola, respectively. Moreover, time–kill assays confirmed the bactericidal activity at different timepoints: 4 h for CFS, 6 h for vitamin C, and 24 h for acerola. The fractional inhibitory concentration index (FICI) showed indifference for all combinations, and for associations tested at 2, 4, and 8XMIC. S. mutans biofilm production was impaired for all components, with stronger activity by vitamin C and acerola at lower concentrations. The probiotic formulation containing L. reuteri SGl01, vitamin C, and acerola extract exerts a bactericidal effect, especially strong for the CFS, as well as antibiofilm activity. Thus, the combination of these three components could be advantageous for their complementary effects, with use as a novel treatment against the development of dental caries by S. mutans.