Background: Antibiotic resistance is becoming an increasingly urgent problem for human and animal health due to the widespread use of antibiotics in medicine, veterinary medicine, and agriculture. At the same time, the natural reservoirs of antibiotic-resistant pathogens remain unclear. Wild birds may play a role in this due to their biology. Escherichia coli is a representative indicator pathogen for antibiotic resistance studies. Materials and Methods: In 2020-2021, sampling of feces and cloacal swabs from six species of wild waterfowl (Eurasian wigeon Anas penelope, Eurasian teal Anas crecca, white-fronted goose Anser albifrons, red-breasted goose Rufibrenta ruficollis, graylag goose Anser anser, shelduck Tadorna tadorna) and from two species of domestic waterfowl (ducks and geese) was conducted in the Kherson, Zaporizhzhia, Odesa, Kharkiv, and Cherkasy regions of Ukraine. Biological material was collected, stored, and transported in cryotubes with transport medium (brain heart infusion broth [BHIB] with the addition of 15% glycerol) in liquid nitrogen. Bacteriological studies were carried out according to standard methods for the isolation and identification of microorganisms. Drug resistance of E. coli was carried out by a standard disk diffusion method. Results: Bacteria representing six families (Enterobacteriaceae, Yersiniaceae, Morganellaceae, Bacillaceae, Pseudomonadaceae, Staphylococcaceae) were isolated from clinically healthy wild birds (wigeon, Eurasian teal, white-fronted goose, red-breasted goose, mallard, graylag goose, shelduck) in the southern regions of Ukraine with isolation rates ranging from 26.7% to 100%. A total of 19 E. coli isolates were cultured from 111 samples from wild birds, and 30 isolates of E. coli were cultured from 32 poultry samples. E. coli was isolated from birds of all species. The prevalence of E. coli ranged from 5.0% to 33.3% in wild waterfowl and from 90.9% to 100% in domestic waterfowl. The prevalence of multidrug-resistant (MDR) E. coli ranged from 10.0% to 31.8% in wild and domestic waterfowl: 3 of 15 (20%) specimens from wild mallard were MDR in the Kherson region, as well as 7 of 22 domestic ducks (31.8%) and 1 of 10 geese (10%) in the Kharkiv and Cherkasy regions. Isolates from wild birds were the most resistant to ampicillin (AMP), amoxiclav (AMC), amoxicillin (AMX), doxycycline (DO), and chloramphenicol (C). Isolates from poultry were resistant to ampicillin, amoxiclav, doxycycline, amoxicillin, chloramphenicol, and enrofloxacin (EX). Most of the other E. coli isolates from wild waterfowl were classified as non-multidrug-resistant (non-MDR) forms. Analysis of antibiotic sensitivity phenotypes showed that only four antibiotic-resistant phenotypes were detected among non-MDR bacteria, whereas among the MDR bacteria, two antibiotic-resistant phenotypes were detected in mallards and six in domestic waterfowl. Conclusion: The results of this study showed that wild waterfowl in Ukraine, which live in natural conditions and do not receive any antimicrobial drugs, are carriers of E. coli that are resistant to a number of antibiotics that are actively used in industrial poultry.