Escherichia coli (E. coli) is a major bacterial pathogen responsible for neonatal diarrhea in calves, leading to significant economic losses in the livestock industry due to decreased productivity and increased veterinary costs. This study aims to isolate and identify E.coli from intestinal infections in calves using microbiological and biochemical tests and vitik. A total of 75 rectal swab samples were collected from diarrheic calves, different ages (2 days to 6 months) as well as different sex (male or female) from several farms in the Babylon district between October 2024 and February 2025. Out of the 75 samples, 70 (93.3%) showed positive bacterial growth, while 5 (6.66%) were negative bacterial growth, the positive growth include Gram Negative bacteria 50(71.4%) isolates were identified as Escherichia coli 30 (60%),Salmonella typhimurium 8 (16%), Klebsiella spp. 6 (12%) , Mycoplasma bovin 4 (8%), Haemophilis somnus 2 (4%) ,while the remaining Gram positive bacteria 20(28.5%) which revealed 8 (32 %) isolates of Staphylococcus spp. 9 (45%), Enterococcus spp. 6 (30%), 3 (15%)Listeria monocytogenes, 2 (10%)Clostridium spp.