Oral cavity harbors human oral microecosystem, one of the largest microbial reservoirs in the human body. The oral microorganisms usually stably adhere to the surfaces of oral mucosa and teeth in the form of biofilm, and the composition and interaction of biofilms are complicated. Under pathological conditions, microbes can penetrate the pulp cavity and alveolar bone to cause common oral infectious diseases such as dental caries, periapical periodontitis, periodontitis. The infection caused by oral biofilms is even closely related to many systemic diseases. Currently, it is still challenging to study the pathogenic mechanism of the oral biofilm. Moreover, under the influence of the host immunity and various physical and chemical factors inside or outside the oral cavity, the phenotypes and biological characteristics of biofilms remain unclear due to lag of technological advances to mimic and study biofilms in vitro. Microfluidics is a multi-disciplinary field that involves flexible control of micro-scale fluids. It can not only accurately simulate the physical and chemical microenvironment but also be used to conduct real-time observation, high-throughput testing, and single-cell analysis. In this article, we will review the recent research progress of microfluidic technologies in biofilm study, and elaborate on the application prospects of microfluidics for the study of microbial biofilms in the oral microecosystem.