Entamoeba gingivalis is a parasitic protozoan found in the mouth of patients suffering from periodontitis, a widespread oral disease with an underestimated prevalence and major consequences on health. We present the development of the first TaqMan PCR assay targeting both E. gingivalis subtypes. This method has been evaluated on 50 samples from patients diagnosed with periodontitis in comparison with 2 different conventional PCRs, and a real-time SYBR Green PCR. Fifty percent of the samples were found positive for the E. gingivalis ST1 subtype with this new PCR, the SYBR Green PCR and one of the conventional PCRs. Among the 25 remaining samples, 12 (24%) were found positive for the E. gingivalis ST2 kamaktlii variant. This new TaqMan PCR could be used before and after periodontitis treatment to follow its efficacy and measure the parasite load in order to better understand the role of these parasites in oral diseases.