Calf diarrhea due to Escherichia coli (E. coli) causes huge economic losses and possess an important veterinary health aspect. In current study, antibiotic profiling, molecular screening, and identification of stx-1, eaeA, escV and bfpA virulence genes of E. coli isolated from calves were carried out. Fecal samples from 28 diarrheagenic calves were taken, processed and cultured on MacConkey's agar, followed by identification using biochemical tests. Antibiotic susceptibility of the isolates was checked by disc diffusion method. For molecular characterization, polymerase chain reaction (PCR) was performed by using specific primers. Out of 28 samples collected from diarrheagenic calves, 83% samples were found positive for E. coli on the basis of biochemical profiling. Antimicrobial susceptibility against various antibiotics exhibited that most of the E. coli isolates were multi-drug resistant. All the isolates showed 100% resistance to rifampicin, erythromycin and oxacillin. The E. coli isolates were not resistant to gentamycin. Analysis through PCR showed that 10% of the isolates were positive for stx-1 gene, however, bfpA, escV and eaeA genes were not detected. Thus, E. coli is one of the key bacterium causing calve diarrhea and it was found that among all the virulent genes the stx- 1plays a part in E. coli associated diarrheal infection in calves. [ABSTRACT FROM AUTHOR]