Subclinical mastitis (SCM) poses a considerable burden and challenge to modern dairy management. The occurence of Escherichia coli (E. coli) in numerous categories of food such as meat, egg, and milk also increases a public health concern, particularly through the consumption of unpasteurized dairy products, impacting human health. The primary objective of current study was to detect E. coli from states of sub-clinical mastitis in buffalo. The researchers employed classical methods for isolation and identification. Additionally, they utilized PCR (Polymerase Chain Reaction) techniques to detect specific virulence factors encoding genes, namely Stx1, and Stx2, which are associated with the pathogenicity of E. coli. Between March and July 2023, a total of fifty milk specimens were taken from buffalo. These specimens were obtained from diverse regions, including Kenitra, Haoy Al-Kanysa, Busyf, Al-Shamseat, Badoush, Kuba, and Tlkeif, all situated in Nineveh province. The findings of the current study indicated the proportion of E. coli detected in the milk was 42% (21 out of 50 specimenss). Additionally, during the molecular monitoring for an exact E. coli gene, it was observed totally isolates (100%) have the uidA gene. One isolate of E. coli possessed the Stx1 gene 1/21 (4.8%). The Stx2 gene was not discovered in E. coli. The results of this research could contribute to addressing a key factor in subclinical mastitis in buffalo, offering potential benefits for the private sector in effectively managing the disease. E. coli isolates carry virulence factors that are responsible for causing food poisoning in humans.