In vivo dental plaque biofilms consist of complex communities of oral bacteria that are a challenge to replicate in vitro. The aim of this investigation was to establish human dental plaque microcosms in microplates to reflect conditions that are relevant to dental caries. Microcosm plaque biofilms were initiated from the saliva of two different donors, grown for up to 10 days in 24-welled microplates on ThermanoxTM coverslips in various types of artificial saliva with and without sucrose, which were replaced daily. Microbiota composition of 40 species associated with oral health and dental caries was monitored in the plaques using Checkerboard DNA–DNA hybridization analysis. pH was measured as an indicator of cariogenic potential. The composition of the saliva inocula was different, and yielded plaque microcosms with different composition and growth responses to sucrose. Artificial saliva type and presence of sucrose, and the resulting growth and pH conditions, modified the growth of individual species and hence the ecological profile of the microplate plaques during development. Complex population shifts were observed during development, and older plaques comprised predominantly facultative anaerobic species. Sucrose supplementation limited the decline of Streptococci over time but did not increase the abundance of mutans Streptococci. Sucrose at 0.15% increased levels of caries-associated species including Lactobacillus fermentum, Lactobacillus acidophilus and Actinomyces gerensceriae; these were further increased with sucrose at 0.5%, in addition to Actinomyces israelii, Rothia dentocariosa and Capnocytophaga gingivalis. The microplate plaques demonstrated complex community dynamics that appeared to reflect the maturation of natural plaques, and sucrose induced a cariogenic plaque composition and pH.