Laser-based Fourier-Transform Raman spectroscopy (FTRS) has been used to identify in situ compounds of ecophysiological significance in diverse field-fresh Antarctic cryptoendolithic microbial communities. FTRS does not disrupt the community and permits characterization of visible and invisible compounds in their natural configuration within cells and their current or former microhabitat. The small “footprint” of the microscopic laser beam permits accurate analysis of discrete zones of compounds produced by extant or degraded micro-organisms with minimum destruction of the biota. This spatial chemical analysis is applicable to any translucent or exposed habitat or biotic assemblage. Two hydrated forms of biodegradative calcium oxalate were differentiated in black-pigmented and hyaline lichen zones of endolithic communities. The oxalate was restricted to zones containing fungi. Communities dominated by cyanobacteria at Battleship Promontory (77°S) and a newly discovered site at Timber Peak (74°S) contrasted chemically with those dominated by eukaryotic algae at East Beacon (78°S). FTRS also showed the zonation of pigments including chlorophyll and UV-protective carotenoids in situ. At extreme sites on the polar plateau, it revealed the presence of “fossil” endolithics where detrimental climatic changes had made the microbes non-viable or amorphous, being represented solely by their residual bio-molecules. The technique has potential for past or present life-detection anywhere in the world without destruction of the microniche.