Aguamiel is a beverage produced by some Agave species that is consumed in its fresh or fermented form. Despite its uses and popularity, seasonal effects on its microbial and chemical profiles are unknown. In this study, using aguamiel collected from A. salmiana and A. atrovirens during different seasons, we identified microorganisms by sequencing the 16S and 18S rDNA genes and determined their chemical profiles. In total, 49 microbial strains were identified (38 bacteria and 11 yeasts). The highest richness and biodiversity were observed during winter and summer. Different lactic acid bacteria and yeast genera with potential industrial applications were identified, such as Acetobacter, Lactobacillus, Leuconostoc, and Clavispora. The analysis of the chemical profiles indicated the presence of maltooligosaccharides and fructooligosaccharides, which are associated with human health improvements, during spring in Agave aguamiel. Aguamiel can be used in the food industry due to its microbiological and chemical profiles.