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 in Gharbia governorate, Egypt. A 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 samples from milk were E. coli O125 (3%) followed by E. coli O157 and E. coli O55 (1% for each), while in worker's urine samples, E. coli O157 was (3.4%) and E. coli O125 was (1.7%). The same E. coli serogroups were found in both bovine milk and workers' urine, posing a serious threat to both human and animal health due to zoonotic transmission of these important serotypes.