Seventy-two fecal samples obtained from wild animals in Portugal were sampled on Levine agar plates (nonsupplemented with antibiotics), and Escherichia coli isolates were recovered from 56 of them (78%), obtaining a total of 112 E. coli isolates (two per sample). Susceptibility to 16 antibiotics was studied in these isolates, and the following percentages of resistance were obtained: tetracycline, streptomycin, ampicillin, and trimethoprim-sulfamethoxazole (SXT) (range 19–35%); nalidixic acid (14%); ciprofloxacin (9%); amoxicillin-clavulanic acid, gentamicin, tobramycin, and chloramphenicol (range 4.5–7%); cefotaxime, and aztreonam (1.8%); ceftazidime (0.9%); and amikacin, cefoxitin, and imipenem (0%). A bla _TEM gene was found in 22 of the 25 ampicillin-resistant isolates, and the gene encoding CTX-M-14 β-lactamase was identified in the two cefotaxime-resistant isolates (recovered from a common kestrel and a sparrowhawk), associated with bla _TEM-52 gene in one of them. Other resistance genes detected were as follows: aac (3)-II or aac (3)-IV genes in all gentamicin-resistant isolates; aadA1 or aadA2 in 22 of 25 streptomycin-resistant isolates; tet (A) and/or tet (B) in all 39 tetracycline-resistant isolates; and sul1 and/or sul2 and/or sul3 genes in all 21 SXT-resistant isolates. Two amino acid changes in GyrA protein (Ser83Leu + Asp87Asn) and one change in ParC protein (Ser80Ile) were identified in all 10 ciprofloxacin-resistant isolates of our series. The intestinal tract of wild animals is a reservoir of antibiotic resistance genes, especially for ampicillin, tetracycline, streptomycin, and SXT, and it is also remarkable that multiresistant E. coli isolates are detected in some of the tested animals.