This chapter discusses the critical roles of microenvironments within oral biofilms in health and disease. Extracellular polymeric substances (EPS) play a central role in shaping the chemical and physical microenvironments that modulate microbial colonization and interactions on various oral soft and hard tissue surfaces. These microenvironments are crucial for maintaining ecological homeostasis, enabling the coexistence of hundreds of species. However, perturbations in these highly balanced environments can lead to dysbiosis and subsequent development of pathogenic biofilms that are associated with oral diseases such as dental caries and periodontal disease. Here, we highlight the complex, multifactorial nature of the biofilm microenvironment, driven by dynamic changes in microbial behavior and EPS characteristics. Furthermore, we discuss the importance of these microenvironments in understanding the pathogenesis of biofilm-mediated oral diseases and in developing targeted therapeutic strategies that can be leveraged to effectively control such diseases.