Apical periodontitis caused by microbial infection in the dental pulp is characterized by inflammation, destruction of the pulpal and periradicular tissues, and alveolar bone resorption. We analyzed the chronological changes in microbiota using a pyrosequencing-based approach combined with radiologic and histopathologic changes in a rat apical periodontitis model. During the three-week observation, the pulp and periapical area showed a typical progress of apical periodontitis. A total of 27 phyla, 645 genera, and 1276 species were identified. The root apex had a lower bacterial species diversity than the pulp chamber. Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria were dominant phyla in both the pulp chamber and root apex. Remarkably, bacterial communities showed a tendency to change in the root apex based on the disease progression. At the genus level,Escherichia, Streptococcus, Lactobacillus, Rodentibacter, andBacteroideteswere dominant genera in the pulp chamber. The most abundant genera in the root apex wereBradyrhizobium, Halomonas, andEscherichia. The speciesAzospirillum oryzaeincreased in the pulp chamber, whereas the speciesBradyrhizobium japonicumandHalomonas stevensiiwere highly observed in the root apex as the disease progressed. The experimental rat model of apical periodontitis demonstrated a relationship between the microbiota and the apical periodontitis progression.