The study aimed to determine the relationship between antibiotic resistance and the ability to produce biofilm of E.coli isolated from sheep in Nineveh Governorate. One hundred four fecal swabs were collected from healthy sheep from 1st February to 5th March 2022. Standard microbiological methods include culture on eosin methylene blue agar (EMB) and MacConkey agar, confirmed by Gram's Stain and biochemical tests, then polymerase chain reaction (PCR) assay for the specific gene of E.coli (uidA). Antibacterial resistance for 12 types of antibiotics and biofilm production tests were done by Congo red agar. The results showed that 92 samples at a rate of 88.46% were positive for the E.coli isolates; the study also showed that 71 isolates at a rate of 77.17% of E.coli isolates could produce biofilm. The study concluded that biofilm-producing E.coli  in both forms strong and weak appear higher resistant to antibiotics other than the non-productive ones. Therefore, searching for other methods to test bacterial sensitivity to antibiotics that consider many factors such as biofilm production and extended-spectrum beta-lactamases is necessary.