Metabolites can provide useful biomarkers to indicate changes in the soil microbial community. Here, we quantitatively assessed potential metabolite markers associated with biocrusts from early successional stages (cyanobacteria) to late successional stages (moss or lichens) in a desert ecosystem via untargeted metabolomics analysis. We identified 570 metabolites in total, several of which varied substantially among the different successional stages, thus serving as potential successional-stage biomarkers. Lipids and lipid-like molecules, as well as several volatile organic compounds (phenylpropanoids and polyketides/benzenoids), may serve as potential biomarkers to identify stages of the cyanobacteria-lichen-moss successional trajectory in biocrusts.