Sotol is an alcoholic beverage obtained after the spontaneous fermentation of the sotol plant (Dasylirion spp), commonly known as a pineapple. However, the microorganisms and chemical changes involved during this fermentation process are unknown. The objectives of this research were to identify the cultivable and non-cultivable microorganisms involved during this fermentation using PCR, PCR-DGGE, and the sequencing of the 16S and 18S ribosomal genes, and to determine the chemical changes that occur throughout the fermentation process. Results showed the presence of 28 microorganisms during the sotol (Dasylirion durangensis) production which corresponded to Bacillus, Lactobacillus, Kluyveromyces and Pichia genera, with the major-species richness, occurring in the dry fermentation stage. On the 7th day of fermentation, 80% of total sugar was consumed. While, on the 10th day of fermentation, the highest (96%) consumption of total sugar and the highest (9.5% v/v) ethanol concentration were observed. During this stage of fermentation, most of the microorganisms isolated belonged to Lactobacillus, Bacillus, and Kluyveromyces genera. Like in the production of mescal and tequila, on the 10th day of fermentation, the presence of organic compounds such as isoamyl acetate, isoamyl alcohol, limonene, and 2-phenylethanol was confirmed. The knowledge gathered about the microorganisms and chemical changes involved during sotol production could help to standardize the process for obtaining a beverage with organoleptic characteristics desirable to consumers.