Escherichia coli O157 is a Shiga toxin-producing E. coli causing disease in humans. Cattle are the primary reservoir of the pathogen. Information regarding the contribution of cattle to diarrheal illnesses in humans through consumption of contaminated beef is scarce in Ethiopia. We collected samples from 240 cattle, 127 beef, and 216 diarrheic patients in Bishoftu town in Ethiopia to assess the occurrence and determine the virulence genes, genetic relatedness, and antimicrobial resistance of E. coli O157. E. coli O157 was detected in 7.1% of the rectal content samples from cattle in slaughterhouses, in 6.3% (n = 127) of the beef samples, and in 2.8% of the diarrheic patients' stool samples. All isolates were positive for eae gene, 24 (77%) of them were positive for stx2 gene (21 stx2c and 3 stx2a), whereas stx1 gene was not detected. Molecular typing grouped the isolates into eight pulsed-field gel electrophoresis pulsotypes with three pulsotypes containing isolates from all three sources, one pulsotype containing one isolate from human origin and one isolate from beef. The remaining four pulsotypes contained isolates unique either to beef or to humans. With the exception of 1 multidrug-resistant isolate from beef, which was resistant to 8 antimicrobial drugs, the remaining 30 isolates were susceptible to the 14 antimicrobials tested. In conclusion, the finding of genetically similar isolates in cattle, beef, and humans may indicate a potential transmission of E. coli O157 from cattle to humans through beef. However, more robust studies are required to confirm this epidemiological link.