The aim of this research was to study the effect of varying incubation times and sucrose exposures on lesion development and biofilm composition using a multi-species biofilm caries model. Two studies were conducted. In study 1, enamel specimens were divided into four groups, inoculated with a mixed overnight culture of Streptococcus mutans, Lactobacillus casei, Actinomyces naeslundii, Streptococcus parasanguis and Streptococcus salivarius, and exposed to circulating trypticase soy broth + 5% (w/v) sucrose (TSBS; 30 min, three times per day (3 × /day)) and a mineral washing solution containing 0.25 p.p.m. fluoride (22.5 h/day) for 2, 5, 6 or 8 days. In study 2, additional enamel specimens were divided into four groups and exposed to the same biofilm for 7 days, but with variations in the feeding schedule: TSBS 3×/day for 5 min, TSBS 3×/day for 15 min, TSBS 3 × /day for 30 min, or TSBS 3×/day for 30 min + 1×/day for 15 min. At the end of each study, bacterial colonization counts and lesion size were determined. In study 1, specimens developed significantly deeper carious lesions with longer demineralization time (average lesion depth was 52.16 μm, 67.86 μm, 84.91 μm, and 99.97 μm, respectively, for 2, 5, 6, and 8 days). In study 2, there was no significant difference in size between the lesions developed at feeding schedules of 3 × /day for 5 or 15 min. Lesions exposed to longer (30 min) and more frequent feeding schedules (4×/day) were significantly larger than the other groups. In both studies, all five bacterial species were able to colonize the enamel and were present in all groups at the end of the experiments, with predominance of lactobacilli over S. mutans. In conclusion, larger lesions were observed with increased incubation time and more frequent feeding schedules, with small variations in biofilm composition.