Cattle producers usually face considerable economic losses due to neonatal Calf diarrhea (NCD). Aiming to delineate this problem, we conducted the bacteriological, serological and molecular studies on 100 fecal swabs collected from diarrheic calves (1 day to 3 months) old from different farms in Garbia Government, Egypt in the time from December 2021 to May 2022. E. coli isolation rate was 34% with the Multi drug resistance being a general trend in the majority of the isolates. Sero-grouping of E. coli resulted in the encountering of O1, O26 , O44 ,O55, O115, O125 , O127, O128 (5% for each type), O18, O119 (10% for each type) and K99 ( 25%). Virulence and resistance genes molecular investigation using PCR in 2 isolates of E.coli serotype(K99) revealed the presence of virulence genes (LT, STa) and Resistence genes (qnrA, aac(6)-IB-cr, blaTEM , KPC , Sul , aadB). We concluded that; the higher stress the E. coli are facing by the improper use of anti-bacterial drugs, the more advanced and newly mechanisms the bacteria develop to overcome this challenge. Therefore the multi-drug resistant strains of E.coli are continuously emerged which impose a serious threats to the human and animal health.