Shiga toxin-producing Escherichia coli (STEC) is one of the most frequent bacterial agents associated with food-borne outbreaks in Europe. In humans, the infection can lead to life-threatening diseases. Domestic and wild animals can harbor STEC, and ruminants are the main STEC reservoirs, although asymptomatic. In the present study we have characterized STEC from wildlife (wild boar (n = 56), red deer (n = 101), red fox (n = 37) and otter (n = 92)). Cultivable STEC (n = 52) were isolated from 17% (n = 49) of the faecal samples. All the isolates were non-O157 STEC encoding stx1 (n = 2; 4%) and/or stx2 genes (n = 51; 98%). Only one strain (2%) isolated from red fox had an antibiotic resistant phenotype. However, when the normalized resistance interpretation of epidemiological cutoffs (NRI ECOFFs) were used, 23% (n = 12) of the strains were non-wildtype to at least one of the antibiotics tested. After analysis by pulsed-field gel electrophoresis (PFGE), 20 strains were selected for whole genome sequencing and belonged to the following serotypes: O27:H30 (n = 15), O146:H28 (n = 2), O146:H21 (n = 1), O178:H19 (n = 1), and O103:H2 (n = 1). In addition to stx, all strains encode several virulence factors such as toxins, adhesins, fimbriae and secretion systems, among others. All sequenced genomes carried several mobile genetic elements (MGEs), such as prophages and/or plasmids. The core genome and the phylogenetic analysis showed close evolutionary relationships between some of the STEC recovered from wildlife and strains of clinical origin, highlighting their pathogenic potential. Overall, our results show the zoonotic potential of STEC strains originating from wildlife, highlighting the importance of monitoring their genomic characteristics following a One Health perspective, in which the health of humans is related to the health of animals, and the environment.