The intensive use of antibiotics in human and veterinary medicine, associated with mechanisms of bacterial genetic transfer, caused a selective pressure that contributed to the dissemination of antimicrobial resistance in different bacteria groups and throughout different ecosystems. Iberian wolf, due to his predatory and wild nature, may serve as an important indicator of environmental contamination with antimicrobial resistant bacteria. The aim of this study was to characterize the diversity of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates within the fecal microbiota of Iberian wolf. Additionally, the identification of other associated resistance genes, phylogenetic groups, and the detection of virulence determinants were also focused on in this study. From 2008 to 2009, 237 fecal samples from Iberian wolf were collected in Portugal. E. coli isolates with TEM-52, SHV-12, CTX-M-1, and CTX-M-14-type ESBLs were detected in 13 of these samples (5.5%). This study reveals the presence of ESBL-producing E. coli isolates, in a wild ecosystem, which could be disseminated through the environment. Moreover, the presence of resistant genes in integrons and the existence of virulence determinants were shown. The association between antibiotic resistance and virulence determinants should be monitored, as it constitutes a serious public health problem.