The microbiota from artisanal cheeses produced in the Amazonian region is evaluated. Samples of artisanal cheeses were obtained from markets in Conceição do Araguaia and Redenção (Pará, Brazil) over rainy and dry seasons, and their biodiversity was assessed by culture-dependent and culture-independent methods. Mean counts of lactic acid bacteria (LAB) in cheeses ranged from 7.32 to 8.84 log CFU/g, for both seasons. Members of genera Lactobacillus, Lactococcus, Weissella, Enterococcus, Pediococcus, and Leuconostoc were predominant. The amplification of the 16S rRNA V6-V9 region, followed by a temporal temperature-gradient gel electrophoresis (TTGE) and sequencing of the TTGE bands revealed important differences in the microbial composition variability between samples from the two seasons and among cheese samples analyzed. TTGE showed the presence of microorganisms that are frequently found in cheese, such as L. lactis subsp. lactis, as well as other non-usual species, such as Macrococcus caseolyticus and Corynebacterium variabile. Moreover, TTGE analysis revealed the presence of microorganisms that have been isolated from other types of foods (Paralactobacillus selangorenses) along with some not usually found in foods, such as Exiguobacterium acetylicum, plus the presence of pathogenic microorganisms (Granulicatella elegans and Aerococcus sanguinicola). The present molecular approaches combined with culture-dependent methods provided a more detailed description of the microbial ecology of traditional cheeses from the Amazonian region in northern Brazil.