In our research, we analyzed the resistance of cephalosporin-resistant E. coli strains to antimicrobial agents. The strains were collected during five years from wild animal species commonly inhabiting Poland. We have identified the type of β-lactamases produced and the multidrug-resistance profile. Most strains (73.8%) had genes encoding ESBL enzymes, mainly CTX-M-1 and TEM. Almost all AmpC-β-lactamase-producing isolates had the blaCMY-2 gene. Almost 70% of the strains tested showed a multi-drug resistance profile. The dominant phenotype was resistance to tetracycline (69.05%), and/or sulfamethoxazole (57.1%). We also found high resistance to quinolones: ciprofloxacin 35.7% and nalidixic acid 52.4%. The phenotypic resistance of the strains was in most cases confirmed by the presence of corresponding genes. Among strains, 26.2% were carriers of plasmid-mediated quinolone resistance genes (PMQR). MLST analysis revealed a large clonal variation of the strains, which was reflected in 28 different sequence types. More than half of the strains (54.7%) were classified into the following sequence complexes: 10, 23, 69, 101, 155, 156, 168, 354, 398, 446, and 648. Only one strain in the studied group was assigned to the ExPEC pathotype and represented sequence type 117. The results of our research have confirmed that isolates obtained from wild animals possess many resistance determinants and sequence types, which are also found in food-producing animals and humans. This reflects the doctrine of “One health”, which clearly indicates that human health is inextricably linked with animal health as well as degree of environmental contamination. We conclude that the resistance and virulence profiles of strains isolated from wildlife animals may be a resultant of various sources encountered by animals, creating a rich and varied mosaic of genes, which is very often unpredictable and not reflected in the correlation between the sequence type and the gene profile of resistance or virulence observed in epidemic clones.