Diarrheagenic Escherichia coli (DEC) are considered one of the major etiological agents of neonatal calf diarrhea (NCD). One hundred calves (2-21 days-old) suffering from diarrhea in Sharkia Governorate, Egypt were used in this study. Six E. coli virulence genes were characterized by multiplex PCR including; heat-stable enterotoxins (STa; 33.3%), heat-labile enterotoxin (LT; 30%), two Shiga toxins: (Stx1; 86.67%), (Stx2; 26.67%) produced from Shiga toxin producing E. Coli (STEC) and two enterotoxigenic E. coli (ETEC) adhesions (F5; 13.3%) and (F41; 16.67%). Each isolate was found to carry one or more virulence genes. Clinicobiochemical examination showed significant (P<0.01) increase in serum total protein, globulin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, blood urea nitrogen (BUN), and potassium levels. Meanwhile; serum albumin, A/G ratio, sodium and chloride levels were significantly (P<0.01) decreased. Multiplex PCR was a useful quick diagnostic tool for characterization and identification of E. coli virulence genes. Shiga toxin genes increase the risk for zoonotic STEC infection. Mixed E. coli infection is strongly associated with the reported cases of diarrhea due to the presence of several virulence genes in some isolates.