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The Atacama desert is amongst the oldest and driest deserts in the world, and its hyperarid core is described as the most barren region imaginable’. In this study, we describe for the first time the endolithic microbial communities composed by eukaryotic microorganisms, such as melanized fungi and algae, colonizing the interior of gypsum crusts collected in the hyperarid zone of the Atacama desert. Melanin pigments present in fungi cell walls have been studied by Raman microspectroscopy. Analyses have been performed directly in the gypsum crust sustaining the colonisation of fungi and algae, on the thin sections and on the cultivated culture of the fungi from Neocatenulostroma genus. Raman spectroscopic signature of melanin has been clearly observed in all the sample types. Two broad bands located at wavenumbers 1605-1580cm(-1) and around 1350cm(-1) have been detected in all the studied samples, with slight variations in the appearance of other bands or shoulders of smaller intensity documenting either the changes in melanin composition or other biomolecules bound to melanin. Blue and green lasers (445, 514 and 532nm) have been shown as favourable excitation for the study of melanins as the longer wavelengths induced adverse effects that mask the Raman signal. Effect of laser power level on the resulting spectra has also been discussed. Algal carotenoid pigment has been detected in isolated regions of the sample by the Raman bands of 1520, 1155 and 1002cm(-1). The strong Raman signal of fungal melanin that has been detected in the host rock from the locality characterized by extreme conditions such as Atacama desert could be considered in the discussion about feasible biomarker molecules; however, low specificity of the broad Raman signature and similarity to the spectrum of amorphous carbon must be also taken into account. Copyright (c) 2017 John Wiley & Sons, Ltd.