Ecological approaches can help to correct oral microbial dysbiosis and drive the advent and persistence of a symbiotic oral microbiome, which benefits long-term dental caries control. The aim of this study was to investigate the impact of the prebiotic Galacto-oligosaccharide (GOS) on the growth of probioticsL. plantarum14,917 and its effect on the inhibitory ability ofL. plantarum14,917 against the growth ofStreptococcus mutansandCandida albicansin an in vitro model. Single-species growth screenings were conducted in TSBYE broth with 1% glucose and 1–5% GOS. Interaction experiments were performed using duo- and multi-species models with inoculation of 105CFU/mLS. mutans, 103CFU/mLC. albicans, and 108CFU/mLL. plantarum14,917 under 1%, 5% GOS or 1% glucose. Viable cells and pH changes were measured. Real-time PCR was utilized to assess expression ofC. albicansandS. mutansvirulence genes. Six replicates were used for each group. Student’st-test, one-way ANOVA, and Kruskal-Wallis were employed to compare the outcomes of different groups. GOS significantly inhibited the growth ofC. albicansandS. mutansin terms of growth quantity and speed when the two strains were grown individually. However, GOS did not affect the growth ofL. plantarum14,917. Moreover, 1% and 5% GOS enhanced the anti-fungal performance ofL. plantarum14,917 in comparison to 1% glucose. GOS as the carbon source resulted in a less acidic environment in theC. albicansandS. mutansduo-species model and multispecies model whereL. plantarum14,917 was added. When GOS was utilized as the carbohydrate substrate,S. mutansandC. albicanshad a significant reduction in the expression of theHWP1,ECE1,atpD, andenogenes (p< 0.05). To our knowledge, this is the first study that reported the ability of GOS to neutralizeS. mutans-C. albicanshigh caries of medium pH and to disrupt virulence gene expression. Moreover, as a prebiotic, GOS augmented the inhibitory ability ofL. plantarumagainstC. albicansin vitro. The current study revealed the anti-caries potential of prebiotics GOS and shed light on novel caries prevention strategies from the perspective of prebiotics and probiotics. These findings provide a rationale for future biofilm or clinical studies to elucidate the effect of GOS on modulating oral microbiota and caries control.