The aim of this study was to evaluate the occurrence of Shiga toxin (stx)-producing Escherichia coli (STEC) in diarrheic newborn calves, as well as the resistance profile of this microorganism against antimicrobials routinely used in veterinary therapy. The antimicrobial profile of Eugenia uniflora against E. coli clinical isolates was also analyzed. Specimens from the recto-anal junction mucosa were investigated by using chromogenic medium and identification of E. coli was done using microbiological methods (Gram staining, indole test, methyl red test, Voges-Proskauer test, citrate test, urease test, and hydrogen sulfide test). The stx1 and stx2 genes corresponding to the STEC pathotype were evaluated by using polymerase chain reaction and electrophoresis. The susceptibility profile to antimicrobial agents commonly used in veterinary therapeutic practice and the antimicrobial effect of lyophilized hydroalcoholic extract of E. uniflora L. leaves against E. coli clinical isolates were evaluated by disk diffusion and microdilution methods. Shiga toxin-positive E. coli was identified in 45% of diarrheic newborn calves (stx1 = 23.2%, stx2 = 4.0%, stx1 + stx2 = 18.2%). The frequency of stx-positive E. coli in the bacterial population was equal to 17.0% (168/990 clinical isolates): 97 (9.8%) stx1-positive E. coli, 12 (1.2%) stx2-positive E. coli, and 59 (6.0%) stx1 + stx2-positive E. coli isolates. All stx-positive E. coli analyzed showed resistance to multiple drugs, that is, from 4 to 10 antimicrobials per clinical isolate (streptomycin, tetracycline, cephalothin, ampicillin, sulfamethoxazole + trimethoprim, nitrofurantoin and nalidixic acid, ciprofloxacin, gentamicin, and chloramphenicol). Effective management measures should be implemented, including clinical and laboratory monitoring, in order to promote animal and worker health and welfare, prevent and control the spread of diseases, and ensure effective treatment of infectious diseases. The E. uniflora L. leaves showed inhibition of microbial growth based on the diameter of halos, ranging from 7.9 to 8.0 mm and 9.9 to 10.1 mm for concentrations of 50 and 150 mg/mL, respectively. This plant displayed bacteriostatic action and a minimum inhibitory concentration of 12.5 mg/mL for all clinical isolates. Its clinical or synergistic effects with antimicrobial agents must be determined from clinical and preclinical trials.