Simple SummaryThis study aimed to evaluate the antimicrobial resistance patterns and biofilm production of clinical isolates ofPseudomonas aeruginosawhich is a pathogenic bacterium that can cause infections in dogs. The results showed widespread resistance to various β-lactam antimicrobials, with amikacin and tobramycin being the only effective aminoglycosides. All isolates carried theoprD gene, which is essential in governing the entry of antibiotics into bacterial cells, and all isolates also carried virulence genes. The study emphasizes the importance of continued monitoring of antimicrobial resistance in veterinary medicine and responsible antibiotic use to prevent multi-drug resistance from emerging. The findings of this study have significant implications for the treatment and prevention ofP. aeruginosainfections in dogs and highlight the need for further research to better understand the mechanisms underlying the emergence of multi-drug resistance.AbstractPseudomonas aeruginosais a pathogenic bacterium that can cause serious infections in both humans and animals, including dogs. Treatment of this bacterium is challenging because some strains have developed multi-drug resistance. This study aimed to evaluate the antimicrobial resistance patterns and biofilm production of clinical isolates ofP. aeruginosaobtained from dogs. The study found that resistance to various β-lactam antimicrobials was widespread, with cefovecin and ceftiofur showing resistance in 74% and 59% of the isolates tested, respectively. Among the aminoglycosides, all strains showed susceptibility to amikacin and tobramycin, while gentamicin resistance was observed in 7% of the tested isolates. Furthermore, all isolates carried theoprD gene, which is essential in governing the entry of antibiotics into bacterial cells. The study also investigated the presence of virulence genes and found that all isolates carriedexoS,exoA,exoT,exoY,aprA,algD, andplcH genes. This study comparedP. aeruginosaresistance patterns worldwide, emphasizing regional understanding and responsible antibiotic use to prevent multi-drug resistance from emerging. In general, the results of this study emphasize the importance of the continued monitoring of antimicrobial resistance in veterinary medicine.