Context The deleterious impacts of cat predation on wildlife have been well documented. Additionally, unowned cats may act as reservoirs of disease important to public and companion animal health and their proclivity for roaming and fighting enables effective disease transmission. Urban environments support the highest human populations and companion animal densities, increasing the potential for disease transmission from unowned cats to people and pets. However, there is little data on the prevalence of pathogens in unowned cat populations. Aims This aim of this research was to establish baseline prevalence data for priority pathogens in an urban population of unowned cats. Methods One hundred unowned cat cadavers were collected from the Brisbane City Council region, Queensland, Australia. Blood and additional organ or tissue samples were collected post-mortem. Diagnostic methods for pathogen detection included use of real-time polymerase-chain reaction, commercially available rapid enzyme-linked-immunosorbent assay, lavage and faecal flotation. Key results Pathogen carriage was found in 79% (95% CI 71, 87%) of sampled cats. In total, 62% (95% CI 52, 72%) of cats showed evidence of co-carriage of two or more pathogenic organisms. The overall prevalence found for pathogens and parasites investigated were: Toxoplasma gondii, 7% (95% CI 2, 12%); Coxiella burnetii, 0.0% (95% CI 0, 0%); feline immunodeficiency virus, 12% (95% CI 6, 18%); feline leukaemia virus, 0.0% (95% CI 0, 0%); and gastrointestinal parasites, 76.8% (95% CI 68, 85%). Conclusions This study reports contemporary prevalence data for these pathogens that have not previously been available for unowned cats of south-east Queensland. High rates of gastrointestinal parasitism observed throughout the study population prompt concerns of a general increase in pathogenic prevalence, especially in comparison with that of owned domestic cats, as per previously published literature. The presence of signs of fighting is an important risk factor for increased likelihood of infection. Implications Data produced from this study contribute to informing cat management efforts throughout urban regions. Continued and expanded investigations, considering prevalence and risk factors of pathogens important to human and companion animal health, are recommended for the south-east Queensland area and beyond.