Introduction: Epidemiological investigations have demonstrated a strong link between periodontal disease and stroke. However, relevant microbiological researches are still lacking at present. This study aimed to investigate the microbial composition of dental plaque, blood and cerebral thrombus in patients with acute ischemic stroke (AIS). Hypothesis: We hypothesized that there was a possible translocation for oral bacteria from periodontal pocket into blood and subsequently involving in the process of cerebral thrombus formation. Methods: Thrombus aspirates, dental plaque and venous blood samples were taken from 13 patients (11 males, 65.5 ± 10.7 years) with AIS. Microbiota was analyzed using high-throughput 16S rRNA gene amplicon sequencing and bioinformatics analyses. Results: Proteobacteria was the most predominant phylum in dental plaque, blood and thrombus. There were no statistic difference in alpha (Shannon and Simpson indexes) and beta diversity (weighted UniFrac distance metric) among dental plaque, blood and thrombus, suggesting a high similarity in their microbial compositions. While, an increased trend of species diversity from supragingival plaque, subgingival plaque, blood to thrombus was observed. Besides, several microbes in thrombus were found to be correlated with plasma coagulation levels, including genera Porphyromonas , which is the common cause of periodontal infection. SourceTracker analysis suggested that about 58% microbial population in blood were oral origin and about 75% microbial population in cerebral thrombi were blood origin. Conclusions: This study described the bacterial profile of dental plaque, blood and thrombus in AIS patients. Our study suggested that some of the bacteria in cerebral thrombus might be derived from the blood and oral cavity and there was a close microbial community compositional relationship among these samples.