E. coli O157: H7 is widely recognized as a major foodborne pathogen responsible for diarrhoea, haemorrhagic colitis, and haemolytic-uremic syndrome. This study was carried out in Negele town to determine the prevalence of E. coli O157: H7 in cow flesh, water streams, and soil. A systematic random sampling technique was utilized to gather samples, which were then transported to Bule Hora University's biology laboratory for isolation and identification of E. coli O157: H 7. The colonies identified as E. coli were streaked onto Sorbitol-MacConkey agar and cultured at 37°C for 24 hours. E. coli O157:H7 was verified using a latex agglutination kit. E. coli O157: H7 isolates were tested for antimicrobial susceptibility in vitro against nine different antibiotics. The data was analyzed using SPSS 21 software. The Pearson Chi-square test was used, and differences were judged significant at P ≤.05. Descriptive statistics such as crosstabulation were used to determine the percentage of prevalence in samples. From a total of samples, the overall prevalence of E. coli O157: H7 in cow meat, soils, and streams was determined to be 6.25%, 4%, and 6.66%. The abattoir stores had the highest prevalence of E. coli O157: H7 (3.3%) compared to retailer shops (1.7%). Doxycycline, Ciprofloxacin, and Gentamicin demonstrated the highest susceptibility. Tetracycline was shown to have the highest resistance. Multidrug resistance was shown to be common among isolates. This investigation confirmed a significant prevalence of E. coli O157:H7. Proper training and supervision of meat handlers, farmers, and health care personnel was strongly advised.