In this study, a rare strain of Escherichia coli co-harboring blaCTX-M-32 and blaCTX-M-2 genes was characterized by whole genome sequencing. Escherichia coli strain was recovered from a bovine cecal sample collected at slaughter. The strain was resistant to third and fourth generation cephalosporins, fluoroquinolones, aminoglycosides, tetracycline and sulfamethoxazole, which was in concordance with the genotype: blaCTX-M-32 and blaCTX-M-2, aac(3)-Via and tet(A) and sul1 genes, respectively. The E. coli strain was characterized as pathogenic, and belongs to the ST226 (CC226), serotype O141ab/ac:H5, phylogenetic group A and fumC27/fimH41 types. No virulence genes were found in the genome. According to the plasmid analysis, IncFIB, IncHI2 and p0111 were present. The analysis of the contigs revealed that the blaCTX-M-32 was located in the chromosome, upstream IS5-like/ISKpn26 and IS1380-like/ISEc9 sequences, whereas blaCTX-M-2 was in the p0111 plasmid, within an integron class 1, and flanked by Orf3/qacEΔ1 and IS91/ISCR1. To our knowledge, this is the first report of a multidrug resistant Escherichia coli strain co-harboring two CTX-M enzymes in a healthy bovine from Portugal. This finding allows a better understanding on the dissemination of these resistance mechanism among bacteria of animal origin.