Various bacteria can cause mastitis in cows. Escherichia coli (E. coli) is the most common cause of both clinical and subclinical mastitis on dairy farms, leading to a decrease in milk production and economic losses. Also E. coli is responsible for more than 81% of urinary tract infections (UTIs) in humans and animals, causes dysuria and may lead to kidney damage. The purpose of this study was the detection and serotyping of E .coli isolated from bovines' milk and workers' urine in different dairy farms at Gharbia governorate, Egypt. Bacteriological examination of 158 samples (100 milk and 58 workers' urine) showed that the prevalence of E. coli in bovine milk samples was (5%) and (6.9%) in workers' urine samples. Serological identification of these isolates revealed that the predominant serotypes in isolates from milk were E. coli O125 (60%) followed by E. coli O157, and E. coli O55 (20% for each), while in worker's urin E.coli O157 (50%) and E.coli O125 (25%) .The same E. coli serogroups were detected in both bovines' milk and workers' urine, which pose a significant threat to both human and animal health through the zoonotic transmission of these important serotypes.