Peri-implantitis diseases are characterized by the presence of an inflammatory process that affects the peri-implant tissues under loading, caused by microorganisms and might lead to the implant failure. Investigations on the microbiota of peri-implant biofilm would be important to define effective preventive and therapeutic strategies for peri-implantitis. With the development of sequencing technology, sequencing and analysis technology of 16S rRNA gene have recently been used to evaluate the microbiota associated with osseointegrated implants. These results expanded the knowledge on the diversity of the microbial communities associated with the disease; they also suggested that the microbial structure of peri-implantitis might be different with periodontitis. Here we review the recent progress about the microbiology of peri-implantitis biofilm based on 16S rRNA gene sequence analysis technology.