Light microscopy has enabled the observation of the structure and organisation of biofilms. Typically, the contrast in an image obtained from light microscopy is given by the time-averaged intensity that is effective in visualising the overall structure. Technological advancements in light microscopy have led to the creation of techniques that not only provide a static intensity image of the biofilm, but also enable one to quantify various dynamic physicochemical properties of biomolecules in microbial biofilms. Such light microscopy-based techniques can be grouped into two main classes, those that are based on luminescence and those that are based on scattering. Here, we review the fundamentals and applications of luminescence and scattering-based techniques, specifically, fluorescence lifetime imaging, Förster resonance energy transfer, fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, single-particle tracking, transient state imaging, and Brillouin and Raman microscopy. These techniques provide information about the abundance, interactions and mobility of various molecules in the biofilms and also properties of the local microenvironment at optical resolution. Further, one could use any of these techniques to probe the real-time changes in these physical parameters upon the addition of external agents or at different stages during the growth of biofilms.