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The ESKAPE pathogen, Pseudomonas aeruginosa, poses a serious threat to medical, veterinary, and agricultural practices globally, and thus has been declared a “Priority Pathogen” by the World Health Organisation. Understanding P. aeruginosa prevalence in wild bird populations, livestock, and domestic animals is vital for evaluating potential infection reservoirs. In this study, we screened 1,669 DNA samples obtained between 2010 and 2023 from healthy and diseased wild birds (n = 1,101), domestic animals (n = 269), livestock (n = 133), kangaroos (n = 39), and koalas (n = 127) from Southeast Queensland, Australia, for both P. aeruginosa and overall bacterial load using an ecfX-16 S rRNA duplex real-time PCR assay. P. aeruginosa-positive samples were also screened for the two most common fluoroquinolone resistance genotypes, GyrA Thr83Ile and GyrA Asp87Asn. Overall, only 1.8% of samples from our large and diverse sample set tested positive for P. aeruginosa. Livestock samples showed the highest P. aeruginosa prevalence (4.5%, n = 6), driven primarily by horses (7.4%, n = 5), with wild birds (1.5%, n = 17), koalas (1.6%, n = 2), and domestic animals (1.9%, n = 5) having the next highest rates. In contrast, no P. aeruginosa positive samples were identified in cattle (n = 45) or kangaroos (n = 39). Nearly all positive wild bird samples originated from eye swabs (94%, n = 16). No additional correlation between swab site, health status, or admission cause was identified. No GyrA Asp87Asn variants were seen; however, the GyrA Thr83Ile variant was seen in 2/30 (6.6%) P. aeruginosa-positive samples, both of horse origin. This finding suggests the presence of a fluoroquinolone resistant subpopulation, however confirmation through phenotypic resistance profiling was unable to be performed. Our findings provide important insight into the epidemiology of P. aeruginosa in Australian wildlife and domestic animal populations from South-East Queensland. Further prevalence studies, particularly covering a broader geographical region, are warranted to better elucidate nationwide P. aeruginosa carriage, infection, and fluoroquinolone resistance rates.