Artisanal cheese, produced with raw milk by a predominantly manual approach, has a historical and cultural tradition related to the region of origin. Given its economic and cultural importance, the main objective of this study was to investigate and characterize the diversity of lactic acid bacteria (LAB) of artisanal cheeses produced and traded by family agro-industries in a region of southern Brazil. The LAB composition of artisanal cheese samples, belonging to different municipalities of the Region of Vale do Taquari, were characterized by the next-generation sequencing (NGS) method, amplifying the V3/V4 region of the 16S rRNA gene. A total of 35 LAB species, distributed in seven genera, were identified, and rarefaction analysis suggested that the total diversity assessed by 16S rRNA analysis was high in the analyzed samples. The average Ph ranged from 4.6 to 6.6, and a correlation with the genus Lactococcus (r = 0.62) was the most expressive. The LAB genera identified in the cheese samples were Bavariicococcus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Marinillactibacillus, and Pediococcus. Lactococcus lactis was the most predominant species, present in all samples. Although some species have been identified in the three altitudes studied, the abundance varied according to geographic environments. Enterococcus italicus is more present at high altitudes, unlike Lactococcus plantarum and Lactococcus raffinolactis at low altitudes. Lactococcus lactis was present in the three geographic environments evaluated, but the highest abundance was observed at high altitudes. The identification of LAB present in fermented cheeses is essential to understand the organoleptic quality during the maturation process as well as to establish the shelf life, including the safety and the overall quality of the cheese. This specific microbiota contributes to the flavor and unique characteristics of the regional dairy products, and on the other hand can be a source of specific starter cultures that guarantee the product’s identity.