The origin of human and calf infections by Shigatoxigenic (STEC) and enteropathogenic (EPEC) Escherichia coli O80:H2 is still unknown. The aim of this study was to identify E. coli O80 in healthy cattle with an emphasis on melibiose non-fermenting E. coli O80:H2. Faecal materials collected from 149 bulls at 1 slaughterhouse and 194 cows on 9 farms were tested with O80 antigen-encoding gene PCR after overnight growth in enrichment broths. The 53 O80 PCR-positive broths were streaked on different (semi-)selective agar plates. Five E. coli colonies from 3 bulls and 11 from 2 cows tested positive with the O80 PCR, but no melibiose non-fermenting E. coli was isolated. However, these 16 E. coli O80 were negative with PCR targeting the fliCH2, eae, stx1, stx2 and hlyF genes and were identified by WGS to serotypes and sequence types O80:H6/ST8619 and O80:H45/ST4175. They were phylogenetically related to E. coli O80:H6 and O80:H45 isolated from different animal species in different countries, respectively, but neither to STEC and EPEC O80:H2/ST301, nor to other serotypes of the clonal complex 165. As a conclusion, healthy adult cattle were not identified as a source of contamination of humans and calves by STEC or EPEC O80:H2.