Enterohemorrhagic Escherichia coli (O157:H7) is the primary cause of bloody diarrhea or hemorrhagic colitis. The study was carried out with to determine the epidemiology and antimicrobial resistance pattern of E. coli O157:H7 along the dairy supply chains in Akaki Kaliti sub-city of Addis Ababa, Bishoftu and Sululta towns of central Ethiopia. A cross-sectional study design with random sampling methods was employed. Thus, a total of 450 raw cow milk (294), milker hand swab (65) and water (91) samples were collected from dairy farms, milk collection centers and Cafeterias and processed according to the standards to isolate and identify E. coli O157:H7. The samples were initially enriched in buffered peptone water, then plated onto Sorbitol MacConkey agar. Consequently, the suspected non-sorbitol fermenting colonies were confirmed as E. coli biochemically and serological test using latex agglutination tests. Out of the total 450 samples examined, 6.0% were found to be contaminated by E. coli O157:H7. Accordingly, 9.89% of water, 9.23% of milker hand swab and 4.08% of raw milk samples were contaminated by the pathogen. Furthermore, the prevalence of E. coli O157:H7 was 7.79%, 6.21% and 3.97% in Akaki kaliti sub-city, Sululta and Bishoftu towns, respectively. The result of Fisher exact analysis revealed a significant difference observed (p < 0.05) between the occurrence of the pathogen and the source of sample, sources of water used, sampled material and type of containers. The study also revealed that varying level of resistance of E. coli O157:H7 isolates against nine antimicrobial discs tested and 100% (n = 27) of the isolates showed multidrug-resistance comprising from two up to seven antimicrobial drugs. In conclusion, this study has indicated the occurrence of E. coli O157:H7 and its multiple drug-resistant profiles in milk samples along the dairy supply chains and its risk to public health and food safety. Therefore, proper hygienic practices from dairy farms to fork and rational drug usage are recommended. © 2022 Dejene et al.