Streptococcus mutans is a known key contributor in dental caries development and flourishes in the presence of common dietary sugars such as sucrose. S. mutans forms biofilms on tooth surfaces, facilitating enamel demineralization and promoting dental caries development. This study examines the inhibitory effects of xylitol, a non-fermentable sugar alcohol, on biofilm formation. While xylitol is known to impede biofilm formation due to S. mutans’ inability to metabolize it, this study aims to focus particularly on its potential inhibitory effects on S. mutans biofilm growth even in sucrose-rich conditions. Using extracted human teeth immersed in mucin-enriched artificial saliva as an artificial oral environment, biofilm formation was assessed using a crystal violet plaque assay, with quantification performed via spectrophotometric methods. The experimental conditions included 1% sucrose, 1% xylitol, and a 1% sucrose-1% xylitol mixture. The results reveal statistically significant differences in biofilm accumulation between treatments, with pronounced biofilm accumulation observed in sucrose conditions and minimal biofilm accumulation in xylitol conditions. Most notably, the sucrose-xylitol mixture treatment exhibited lower levels of biofilm accumulation with significantly attenuated growth compared to sucrose alone, implying a potential antagonistic interaction between the two sugars. These findings highlight the potential of xylitol as a preventative agent for dental caries, even under cariogenic conditions. Furthermore, this study contributes to the growing amount of evidence supporting the use of xylitol in oral health strategies, suggesting its potential to be integrated into dental care regimens aimed at reducing caries risk and promoting oral health. Future research should focus on exploring the biochemical mechanisms underlying xylitol’s inhibitory effects and its potential antagonistic interactions with other common sugars and cariogenic bacteria.