Understanding the limiting factors and mechanisms of biofilm processes requires the direct measurement of microscale gradients using the appropriate tools. Microscale measurements can provide mechanistic information that cannot be obtained from bulk-scale measurements. Among the most used and trusted tools in microscale biofilm research are microsensors. The goal of this chapter is to introduce microsensor technology along with several examples to illustrate microscale processes in biofilms that are usually absent in bulk. We define a microsensor for biofilm research as a needle-type sensor with tip diameter of a few microns and a length up to several hundred microns. Microsensors can be used noninvasively to monitor in situ biofilm processes. Both optical and electrochemical microsensors can be used for biofilm applications. Because of newly discovered biofilm processes, the design and use of microsensors require customization and carefully designed experiments. In this chapter we present several examples describing the use of microsensors (1) in environmental biofilms, (2) in medical biofilms, and (3) in biofilms for energy and bioproducts. Microsensors can be the most useful if the measured profiles are integrated into the study of overall biofilm processes.