Confocal laser scanning microscopy (CLSM) became in last years an invaluable technique to study biofilms since it enables researchers to explore noninvasively the dynamic architecture and the reactivity of these biological edifices. The constant development of fluorescent markers and genetic tools along with the improvement of spatial, spectral, and temporal resolution of imaging facilities offers new opportunities to better decipher microbial biofilm properties. In this contribution, we proposed to describe the contribution of CLSM to the study of biofilm architecture and reactivity throughout two different illustrative approaches.