The aim of the present study was to carry out molecular epidemiological investigation on enterotoxigenic Escherichia coli (ETEC) K99 and Salmonella spp. in diarrheic neonatal calves. Fecal samples were obtained from 220 diarrheic calves at 9 farms related to four governorates in central and northern Egypt. E. coli and Salmonella spp. isolates were examined for E. coli K99 and Salmonella spp. using PCR. ETEC K99 was recovered from 20 (10.36 %) out of 193 isolates, whereas Salmonella spp. was recovered from nine calves (4.09%). Multivariable logistic regression was used to evaluate the risk factors associated with both infections. ETEC K99 was significantly affected by age (P<0.01; OR: 1.812; CI 95%: 0.566-1.769), colostrum feeding practice (P<0.01; OR: 5.525; CI 95%: 2.025-15.076), rotavirus infection (P<0.001; OR: 2.220; CI 95%: 0.273-1.251), vaccination of pregnant dams with combined vaccine against rotavirus, coronavirus and E. coli (K99) (P<0.001; OR: 4.753; CI 95%: 2.124-10.641), and vitamin E and selenium administration to the pregnant dam (P<0.01; OR: 3.933; CI 95%: 0.703-1.248). Infection with Salmonella spp. was found to be significantly affected by the animal age (P<0.05; OR: 0.376; CI 95%: 0.511-1.369), Hygiene (P<0.05; OR: 0.628; CI 95%: 1.729-5.612), and region (P<0. 01; OR: 0.970; CI 95%: 0.841-1.624). The results of the present study indicate the importance of PCR as rapid, effective and reliable tool for screening of ETEC and Salmonella spp. when confronted with cases of undifferentiated calf diarrhea. Moreover, identification of the risk factors associated with the spreading of bacteria causing diarrhea may be helpful for construction of suitable methods for prevention and control.