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The rocks of the McMurdo Dry Valleys desert in Antarctica harbour endolithic communities of micro-organisms such as lichens, fungi, cyanobacteria and bacteria. Establishing the physiological status and viability of these microbial colonies in their natural microhabitat has far-reaching implications for understanding the microbial ecology of the harsh environment of this polar desert. Here we describe the use of confocal microscopy and a specific fluorescent probe (FUN-1) to evaluate the metabolic activity of fungal cells. Application of confocal microscopy also served to identify living and dead bacteria or cyanobacteria using the fluorescent assay reagents Live/Dead SYTO 9 and propidium iodide or SYTOX Green, respectively. In addition, through the use of epifluorescence microscopy, live/dead bacteria and cyanobacteria could be detected by estimating fluorescence from their cell components provoked by simultaneously staining with nucleic acids stains such as DAPI and SYTOX Green.