Lake Pastos Grandes in Bolivia is mainly composed of salt flats, which are sporadically and only partially submerged during the wet season. In the present study, the chemical composition of water samples of the lake and some influent rivers was determined. We found that it is likely that the lake was influenced by the dilution of metals from ancient evaporites. We performed the first metagenomic studies of this lake. Analyses of shotgun metagenomics revealed that the relative abundances of Burkholderiales and Pseudomonadales were noteworthy in the water samples, whereas the archaea belonging to the Halobacteriales and Cyanobacteria from subsection III had high abundances in the salt flat. The eukaryotes Crustacea and Diatomea exhibited the highest abundances in the water samples. We investigated further the potential effect of human activities on the nitrogen cycle mobilization in the lake and the propagation of antimicrobial resistance genes. This is the first report about the cycle in the lake. Additionally, rifamycin resistance genes and genes related to efflux pumps, which are not considered a hazard when identified in metagenomes, had the uppermost relative abundances in all sampling points. We found that Lake Pastos Grandes hitherto does not show an appreciable influence by anthropogenic actions. The microbiome of Lake Pastos Grandes, including microbial distribution, the nitrogen cycle and antibiotic resistance genes, was analyzed.