Sediments, microbial mats, biofilms and other microbial communities are characterized by steep gradients of physical and chemical parameters. Microsensors are powerful tools to measure these parameters with a sufficient spatial resolution and with a small disturbance of the micro-environment in natural systems. Recently, fiber-optical microsensors have been introduced in the field of aquatic biology as an alternative to existing electrochemical microsensors. Such micro-optodes have already been developed for high-resolution measurement of dissolved oxygen and for temperature measurements. They are easy to fabricate and show an improved long-term and storage stability. An overview is given on the development and characterization of different types of micro-optodes for oxygen and temperature. A luminescence lifetime-based device has been developed which is portable and enables microsensing both in the laboratory and under field conditions. Limitations in practical work with optical microsensors are demonstrated, and strategies to overcome them briefly discussed. A micro-optode array as well as a method for high-resolution oxygen imaging in sediments are presented as two different ways to investigate the two-dimensional oxygen distribution in heterogeneous living systems. Future applications and developments in micro-optode research will be discussed briefly.