Background and Aim: Escherichia coli O157:H7 is enterohemorrhagic E. coli, which produces verocytotoxin or Shiga toxin. It is a well-known cause of severe diseases in humans worldwide. Cattle and other ruminants are the main reservoirs of this organism. Sports animals, such as fighting bulls, riding horses, and fighting cocks, are economic animals in Southern Thailand. This study aimed to identify E. coli O157:H7 from the rectal swabs of these sports animals and determine the antimicrobial susceptibility patterns of isolated bacteria. Materials and Methods: The rectal swabs were collected from 34 fighting bulls, 32 riding horses, and 31 fighting cocks. The swabs were cultured on MacConkey (MAC) Agar; the suspected colonies were then identified by VITEK^® 2 GN card, and the antimicrobial susceptibility was tested by VITEK^® 2 AST N194 in VITEK^® 2 Compact automation. Escherichia coli O157:H7 was confirmed by culturing on sorbitol MAC agar, the ability to grow at 44°C, and the presence of H7 antigen. In addition, the eaeA (E. coli attaching and effacing), along with stx1 and stx2 (Shiga cytotoxins) genes, were determined using polymerase chain reaction. Finally, the cytotoxicity of Shiga toxin was confirmed using the Vero cytotoxicity test. Results: Fifty-five suspected isolates (56.70%), which were collected from 19 fighting bulls (55.88%), 13 riding horses (40.63%), and 23 fighting cocks (71.13%), were identified as E. coli. However, one sample (Bull H9/1) from fighting bulls had an equal confidence level (50%) for E. coli and E. coli O157. The confirmation of this isolate demonstrated that it was sorbitol non-fermenter, could assimilate L-lactate, was unable to grow well at 44°C, and reacted with anti-serum to H7 antigen. In addition, it was positive with stx2 and eaeA genes, and the toxin affected Vero cells by a dose-dependent response. The antimicrobial susceptibility test revealed that five out of 55 (9.09%) E. coli isolates were resistant to antimicrobial agents. All five isolates (21.74%) were collected from fighting cocks. Escherichia coli Cock H4/3 was only one of the five isolates resistant to three antimicrobial agents (ciprofloxacin, moxifloxacin, and trimethoprim/sulfamethoxazole). Fortunately, it was not multidrug-resistant bacteria. Conclusion: This is the first report on detection of E. coli O157:H7 in fighting bulls and antibiotic-resistant characteristic of E. coli in fighting cocks in Southern Thailand. This research is beneficial in preventing the dissemination of E. coli O157:H7 or antimicrobial agent-resistant E. coli in sports animals and humans. © 2022 Veterinary World. All rights reserved.