Faecal samples of 2660 domestic animals from 116 farms and 956 samples of food were examined for the presence of Shiga toxin-producing Escherichia coli (STEC). STEC was recovered from 126 (15·3%) cattle, 135 (11·3%) pigs, 135 (66·8%) sheep, 31 (73·8%) goats, 4 (1%) chicken, and 15 (1·6%) food samples. Of all STEC isolates, 21·5, 25·8 and 15% produced enterohaemolysin, α-haemolysin, and aerobactin respectively, 1·6% displayed localized adherence (LA) to HEp-2 cells, 27·6% were sorbitol negative, and 30% were resistant to antibiotics. Only 14 (3·1%) of the STEC isolates belonged to human infection-associated serogroups (O26, O55, O111, O128 and O157), designated as enterohaemorrhagic E. coli (EHEC). This study revealed that STEC are prevalent in domestic animals, and to a lesser extent in food of animal origin in Serbia, but the absence of a EHEC phenotypic profile (characteristic serogroup, LA, enterohaemolysin production) in most animal STEC strains may explain the low incidence of human STEC infection in this part of the world.