Neonatal Calf diarrhea(NCD) is a commonly reported disease and considered as a major cause of economic loss to cattle producers. A total of 100 fecal swabs collected from diarrheic calves(0 day-2monthes) old during the period from December 2015 till January 2017 and subjected for bacteriological ,serological and molecular investigations. The infection rate of E.coli was 47% followed by Pseudomonas aeruginosa( 4% )and Salmonella Typhimurum( 1%). Multi drug resistant appeared in the most tested microorganisms. Serogrouping of E. coli revealed the presence of O142 ,O55 ,O11,O27, O157,O119 ,O26 and O127 by a percentange of 20.69%,17.24%,13.8%,6.9%,6.9%.6.9%,3.45% and 3.45%, respectively. Multiplex PCR was applied for detection of virulence genes stx1(5/10),stx2(3/10) and eae(6/10)) which detected in E.coli and stn gene in Salmonella typhimurum and also (blaVIM( 3/4), mexR(4/4)) genes for Pseudomonas aeruginosa were detected