Physicochemical and microbial analyses were conducted for artisanal and semi-industrial manufacturing processes of the Cotija cheese. Differences between manufacturing processes in Cotija cheese affect the microbial composition in the final product. Lactic acid bacteria are important microorganisms in the artisanal Cotija cheese manufacturing. Thirty-one different microorganisms were isolated and identified by ribosomal sequencing analysis during the production of this artisanal cheese. The yeast isolates comprised the following species: Kluyveromyces lactis, Kluyveromyces marxianus, Pichia guillermondii, Rhodotorula mucilaginosa, Galactomyces reessii, and Galactomyces geotrichum. Bacterial isolates were members of the Lactobacillaceae family with the follow species: Pediococcus pentosaceus, Lactobacillus brevis, Lactobacillus plantarum, and Lactobacillus casei. The possible role of these microorganisms in the final flavor and taste, and their utility in the control of undesirable microorganisms in artisanal Cotija cheese is discussed.