The objective of the study was to analyse Streptococcus mutans biofilms grown under different dietary conditions by using multifaceted methodological approaches to gain deeper insight into the cariogenic impact of carbohydrates. S. mutans biofilms were generated during a period of 24 h in the following media: Schaedler broth as a control medium containing endogenous glucose, Schaedler broth with an additional 5% sucrose, and Schaedler broth supplemented with 1% xylitol. The confocal laser scanning microscopy(CLSM)-based analyses of the microbial vitality, respiratory activity(5-cyano-2,3-ditolyl tetrazolium chloride, CTC) and production of extracellular polysaccharides(EPS) were performed separately in the inner, middle and outer biofilm layers. In addition to the microbiological sample testing, the glucose/sucrose consumption of the biofilm bacteria was quantified, and the expression of glucosyltransferases and other biofilm-associated genes was investigated. Xylitol exposure did not inhibit the viability of S. mutans biofilms, as monitored by the following experimental parameters: culture growth, vitality, CTC activity and EPS production. However,xylitol exposure caused a difference in gene expression compared to the control. Gtf C was upregulated only in the presence of xylitol.Under xylitol exposure, gtf B was upregulated by a factor of 6, while under sucrose exposure, it was upregulated by a factor of three.Compared with glucose and xylitol, sucrose increased cell vitality in all biofilm layers. In all nutrient media, the intrinsic glucose was almost completely consumed by the cells of the S. mutans biofilm within 24 h. After 24 h of biofilm formation, the multiparametric measurements showed that xylitol in the presence of glucose caused predominantly genotypic differences but did not induce metabolic differences compared to the control. Thus, the availability of dietary carbohydrates in either a pure or combined form seems to affect the cariogenic potential of S. mutans biofilms. The objective of the study was to analyze Streptococcus mutans biofilms grown under different dietary conditions by using multifaceted methodological approaches to gain deeper insight into the cariogenic impact of carbohydrates. S. mutans biofilms were generated during a period of 24 h in the following media: Schaedler broth as a control medium containing endogenous glucose, Schaedler broth with an additional 5% sucrose, and Schaedler broth supplemented with 1% xylitol. The confocal laser scanning microscopy (CLSM) -based analyzes of the microbial vitality, respiratory activity (5-cyano- 2,3-ditolyl tetrazolium chloride, CTC) and production of extracellular polysaccharides (EPS) were performed separately in the inner, middle and outer biofilm layers. In addition to the microbiological sample testing, the glucose / sucrose consumption of the biofilm bacteria was quantified , and the expression of glucosyltransferases and other biofilm-associated genes was investigated. Xylitol exposure did not inhibit the viability of S. mutans biofilms, as monitored by the following experimental parameters: culture growth, vitality, CTC activity and EPS production. However, xylitol exposure caused a difference in gene expression compared to the control. Gtf C was upregulated only in the presence of xylitol.Under xylitol exposure, gtf B was upregulated by a factor of 6, while under sucrose exposure, it was upregulated by a factor of three. Compared with glucose and xylitol, sucrose increased cell vitality in all biofilm layers. In all nutrient media, the intrinsic glucose was almost completely consumed by the cells of the S. mutans biofilm within 24 h. After 24 h of biofilm formation, the multiparametric measurements showed that xylitol in the presence of glucose caused predominantly genotypic differences but did not induce metabolic differences compared to the control. Thus, the availability of dietary carbohydrates in either a pure or combined form seems to affect the cariogenic potential ofS. mutans biofilms.