Biofilms, which are aggregates of microorganisms and extracellular matrices, widely colonize natural water bodies, wastewater treatment systems, and body tissues, and have vital roles in water purification, biofouling, and infectious diseases. Recently, multiple imaging modalities have been developed to visualize the morphological structure and material distribution within biofilms and to probe the microprocesses in biofilm matrices, including biofilm formation, transfer and metabolism of substrates, and cell–cell communication. These technologies have improved our understanding of biofilm control and the fates of substrates in biofilms. In this review, we describe the principles of various imaging techniques and discuss the advantages and limitations of each approach to characterizing microprocesses in biofilm matrices.