Within Ethiopia, there is a lack of information on the genetic relatedness of from cattle, beef, and diarrheic patients and its potential transmission from cattle to humans through consumption of contaminated beef. The objective of this study was to assess the prevalence and determine the serotypes, genetic relatedness, and antimicrobial resistance of in cattle in two local slaughterhouses, in beef at retail shops, and in diarrheic patients in the only hospital in Bishoftu, Ethiopia. was detected in 2.5% (6/240) of cattle samples, in 8.7% (11/127) of beef samples, and in 2.3% (5/216) of the diarrheic patients. Four serotypes: Typhimurium, Eastbourne, Saintpaul, and Cotham were identified. Typhimurium and Eastbourne were isolated from cattle and beef, whereas Saintpaul and Cotham were isolated only from diarrheic patients. Except for serotype Saintpaul, all isolates were grouped into five pulsotypes, of which two pulsotypes contained isolates from cattle and beef. Isolates from humans represented unique pulsotypes. Among the 22 isolates tested, 95.5% were resistant to at least 1 of the 14 antimicrobials tested. Three isolates originating from cattle were multidrug resistant. One human isolate was susceptible to all antimicrobials tested. More specifically, resistance to ampicillin, sulfamethoxazole, tetracycline, tigecycline, and trimethoprim were observed. The most frequently observed resistance was to sulfamethoxazole (90.9%, 20/22) followed by trimethoprim (22.7%, 5/22). The study revealed considerable contamination of beef at retail shops, antimicrobial resistance to commonly used antimicrobials, and shared genetically similar serotypes between cattle and beef; the link with humans could not be established. Still, the findings of in cattle and beef, the propensity of transfer of from cattle to beef coupled with the common consumption of raw/undercooked beef are likely to pose public health risk in Ethiopia.