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The emergence of bacterial resistance to antibiotic use in veterinary practice is considered a source of multidrug-resistant bacterial infections for humans. Although increasing incidence of antimicrobial resistance in small-animal practices has already been noted, limited information is available about the problem in domestic canaries (Serinus canaria domestica). This cross-sectional study describes the prevalence of Gram-negative bacteria among canaries exhibiting clinical disease signs and the antimicrobial sensitivity patterns of the bacterial isolates. During the breeding season, a bacteriological survey was carried out in 50 amateur breeding aviaries from the Messina Province (Sicily, South of Italy) to detect the prevalence of Gram-negative bacteria in sick birds. Fecal samples from breeder canaries were submitted for bacteriological examination. Of 50 breeding aviaries, 43 (86%) were positive for Gram-negative bacteria. Overall, 88 bacterial isolates, representing 12 genera of bacteria, were cultured. The most frequently recovered bacterial species was Escherichia coli (31/88 isolates, 35.2%). Other frequently isolated species were Enterobacter cloacae (9/88 isolates, 10.2%) and Proteus mirabilis (6/88 isolates, 6.8%). Potentially pathogenic species, including Salmonella Typhimurium (n = 5 strains), Enterobacter sakazakii (n = 4 strains), and Pseudomonas aeruginosa (n = 6 strains), were also identified. The 88 isolates displayed significant frequencies of antibiotic resistance. These results confirm the potential presence of multidrug-resistant bacteria in canary facilities, suggesting that measures to educate the public about this risk are necessary.