The production of cheeses from unpasteurized milk is a public health problem, due to the use of raw milk and the associated poor hygienic conditions. Contamination may occur from several sources, involving several different pathogenic microorganisms, including Escherichia coli. The use of antimicrobials in animals has led to emergence of resistant microorganisms, contributing to the ineffectiveness of these products. The purpose of the current study was to investigate the presence of antimicrobial resistance in E. coli isolates in raw milk cheese in Brazil and to identify the potential risk to public health. A total of 83 cheeses from 3 different cities, Uberaba, Minas Gerais (30), Ribeirão Preto, São Paulo (22) and Aracaju, Sergipe (31) were cultured. From each cheese, 5 colonies were examined and a total of 169 E. coli isolates, 51, 25, and 93 from Uberaba, Ribeirão Preto, and Aracajú respectively were obtained. Ninety-five randomly selected isolates were tested for susceptibility to the 15 antimicrobials: amoxicillin + clavulanic acid (20 + 10 µg), ceftiofur (30 µg), ceftriaxone (30 µg), ciprofloxacin (5 µg), amikacin (30ug), ampicillin (10 µg), cefoxitin (30 µg), gentamicin (10 µg), kanamycin (30 µg), nalidixic acid (30 µg), streptomycin (10 µg), tetracycline (30 µg), chloramphenicol (30 µg), sulfisoxazole (0.25 mg) and trimethoprim + sulfamethoxazole (1.25 + 23.75 µg) used for testing generic E. coli in the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS), by the disk-diffusion (Kirby-Bauer) method. The greatest number of isolates with a resistance pattern (one antimicrobial class or more) was observed in Uberaba, most being resistant to 1–2 antimicrobial classes. Multidrug resistance (resistance to 3 or more classes of antimicrobial agents) was much more frequently observed in isolates from Uberaba and Ribeirão Preto (12 to 14%) than in those from Aracaju (2%). The highest prevalence of resistance in all regions was to tetracycline. Strikingly, prevalence of resistance to ampicillin and amoxicillin/clavulanic acid was much higher in Uberaba than in Ribeirão Preto and Aracajú. For example, 58.85% of Uberaba samples were amoxicillin/clavulanic acid resistant while Ribeirão Preto and Aracaju were 18.18% and 3.57% respectively. For ampicillin, the prevalence in samples were 61.54%, 27.27% and 14.29% to Uberaba, Ribeirão Preto and Aracaju repectively. All 95 isolates were susceptible to ceftriaxone, amikacin, gentamicin, and chloramphenicol. Resistance to ceftiofur was found in Ribeirão Preto, suggesting the therapeutic use this antimicrobial.For statistics analysis, the sample was considered resistant to the antimicrobial when at least one of the isolates demonstrated resistance and it was used exact chi-squared. So, with the test, P-value was 0.001571 and it showed that the city is not resistant independent, thus they do not have the same resistance. And it showed that Uberaba was different from Ribeirão Preto and Aracaju and Aracaju and Ribeirão did not differ (P < 0.05). The variation of resistance to antimicrobials between regions observed in the present study indicates that the cheese made with unpasteurized milk in Brazil may contain E. coli that can be a risk for public health.