Cow's milk is a whole food product that is sensitive to many bacterial contamination. In this study sixty cow milk samples were collected from farm milk, milk vendor and shops milk on Babylon city from October to December 2023, all milk samples were obtained from three source for bacterial availability analysis. All collected samples are processed in laboratory for estimation viable bacterial count and phenotypic detection of Escherichia by plated them on MacConkey agar, and show greenish metallic sheen on colonies in Eosin Methylene Blue. Consequently, antibiotic resistance profile of isolates done by Kirby-Bauer technique on Muller Hinton agar plats, analysis according to recommended by criteria of Instructions of Clinical Laboratory Standards Institute (CLSI), and biofilm formation detection by staining with methylene blue on tissue plate assay. The results showed detection of 53.2% (n=32) positive of Escherichia coli out of sixty cow milk samples and It was confirmed by Vitek system and indole test; farm milk 13.3 % (n=8) with average count 6.7×107 cfu/ml, milk vendor 23.3 % (n=14) with average count1.8×108 cfu/ml, and shops milk 16.6 % (n=10) average count 1.3×108 cfu/ml, and the susceptibility rate of Escherichia coli were tested in the following order: Ciprofloxacin 10 mcg (25.0%), Ciprofloxacin5 mcg (31.2 %), Ampiclox (28.1%),Ampicillin (31.2 %), Trimethoprim (37.5%), Gentamycin (25.0%), Tobramycin (43.7%), Streptomycin (15.6%), Amikacin (28.1%), Doxycillin (31.2 %),Cefaxitin (28.1%),Ceftazidime (25.0%), and Aztreonam(31.2) %. In conclusion, data showed higher contamination of Cow's milk in Babylon city from different source with Escherichia coli, in which biofilm producers and resistance to many antibiotic were notice, Therefore, we recommend periodic monitoring of producers and sellers of raw milk and its products and imposing strict laws to limit the spread of foodborne illnesses and antibiotic-resistant bacteria.