Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens with significant public health implications. The interplay between humans, domestic animals, wildlife, and environmental reservoirs such as water bodies influences their circulation. This study aimed to evaluate the role of the environment in the dissemination of STEC. Fourteen STEC isolates were recovered from stream water and feces of wild animals collected between 2022 and 2024 in a tourist and wild village-like region in Uruguay. All isolates carried the stx2 gene, and one also harbored stx1 and eae (E. coli O157:H7). The remaining isolates were LEE-negative. Most isolates were susceptible to tested antibiotics. Whole genome sequencing (WGS) revealed a wide diversity of serotypes, sequence types, and virulence gene profiles. Pangenome analysis revealed genomic variability among the analyzed isolates, suggesting the presence of adaptive mechanisms and potential ecological plasticity. The Locus of Adhesion and Autoaggregation (LAA), a pathogenicity island described in some LEE-negative strains, was detected complete in three isolates and partially in three others. Core genome SNP-based phylogenetics and cgMLST analysis (chewBBACA) indicated that the local isolates clustered with strains previously reported from food, livestock, and wildlife across the Americas and Europe, suggesting genetic relatedness. These findings underscore the STEC’s environmental diversity and potential public health risk of STEC circulating in a One Health context. The seasonal detection of the isolates, genomic features, and phylogenetic relationships suggests ongoing interactions between human activity, wildlife, and water sources as key factors in STEC ecology.