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The origin of human and calf infections by Shigatoxigenic (STEC) and enteropathogenic (EPEC) <i>Escherichia coli</i> O80:H2 is still unknown. The aim of this study was to identify <i>E. coli</i> O80 in healthy cattle with an emphasis on melibiose non-fermenting <i>E. coli</i> 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 <i>E. coli</i> colonies from 3 bulls and 11 from 2 cows tested positive with the O80 PCR, but no melibiose non-fermenting <i>E. coli</i> was isolated. However, these 16 <i>E. coli</i> O80 were negative with PCR targeting the <i>fliC_H2</i>, <i>eae</i>, <i>stx1</i>, <i>stx2</i> and <i>hlyF</i> genes and were identified by WGS to serotypes and sequence types O80:H6/ST8619 and O80:H45/ST4175. They were phylogenetically related to <i>E. coli</i> 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.