Aims: Common belief suggests that starch is less cariogenic than sugar; however, the related literature is quite controversial. We aimed to compare cariogenic and microbiological effects of soluble starch in both a standard animal model and an oral biofilm system, and to assess the possible substitution of the animal model. Methods and Results: Six-species biofilms were grown anaerobically on enamel discs in saliva and medium with glucose/sucrose, starch (average molecular weight of 5000, average polymerization grade of 31), or mixtures thereof. After 64.5 h of biofilm formation, the microbiota were quantitated by cultivation and demineralization was measured by quantitative light-induced fluorescence. To assess caries incidence in rats, the same microbiota as in the biofilm experiments were applied. The animals were fed diets containing either glucose, glucose/sucrose, glucose/sucrose/starch or starch alone. Results with both models show that demineralization was significantly smaller with starch than sucrose. Conclusions: The data demonstrate that soluble starch is substantially less cariogenic than glucose/sucrose. Significance and Impact of the Study: By leading to the same scientific evidence as its in vivo counterpart, the described in vitro biofilm system provides an interesting and valuable tool in the quest to reduce experimentation with animals.