In this study we developed a rat model of incipient caries to investigate the short-term effects of antimicrobial photodynamic therapy (aPDT) on oral microbiota regulation and demineralization arrestment. Twenty-nine male rats were submitted to caries induction. Early carious lesion was confirmed by optical coherence tomography (OCT) 5 days after experiment beginning in five animals. The remaining animals (n = 24) were randomly divided into two groups: control (n = 12), animals were untreated; and aPDT (n = 12), animals were treated with 100 μM of methylene blue for 5 min and irradiated by a light emitting diode at λ = 645 ± 30 nm, fluence rate of 480 mW cm−2 and exposure time of 3 min. Bacterial burden was evaluated before, immediately after, 3, 7 and 10 days following treatment, and total number of microaerophilic bacteria was counted. OCT was also used to quantify teeth demineralization. A significant bacterial decrease of about 1.6 log was observed immediately after aPDT. Besides, bacterial load in aPDT group remained lower than control until 10 days post-treatment (P < 0.05) and variation of optical attenuation coefficient before and after aPDT was 15%, corroborating to caries arrestment. Put together, these findings suggest that aPDT was competent to reduce cariogenic bacteria and to avoid further mineral loss.