Diarrhea is a complex syndrome causing colossal economic losses. The objective of the current study was to diagnose Enter-pathogens in children, calves, and broiler chickens by a serological and molecular diagnosis of Rotavirus, evaluate Escherichia coli (E. coli) and Salmonella spp. with bacteriological examination beside conventional diagnosis of Cryptosporidium in addition to studying risk factors affecting these pathogens in Assiut Governorate. From February 2021 to April 2021, a total of 60 samples were collected from the stool of children (n=20), feces of calves (n=20) in addition to liver and pool intestine of broiler chickens (n=20) were examined by Immuno Chromatographic Assay (ICA), Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), microbiological examination and modified Ziehl-Neelsen stain smears. The prevalence of Rotavirus infection by ICA was 55% (11/20), 10% (2/20), and 5% (1/20) of examined children, calves, and broiler chickens, respectively. Two (18.18%) of serologically Rotavirus positive stools of children were positive molecularly by RT-PCR, while serologically positive fecal and pool intestines of calves and broiler chickens, respectively, were negative molecularly. There was no significant variation between Rotavirus, E. coli, and Cryptosporidium infections with gender and age of investigated children and between these enteropathogens with sex, age, and species of examined calves. In addition, there were no significant differences between Rotavirus, E. coli, Salmonella, and Cryptosporidium with the age of investigated broiler chickens. It is concluded that these enteropathogens in livestock and chicken living close to the human population necessitate better surveillance and control measures to protect vulnerable animal and human populations.