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Possums and gliders represent a diverse group of marsupial mammals native to Australia, including multiple vulnerable and endangered species. Common brushtail (Trichosurus vulpecula) and common ringtail (Pseudocheirus peregrinus) possums are two urban adapted species frequently found in gardens and parks throughout eastern Australia. Their presence in urban areas provides opportunities for transfer of zoonotic pathogens through both direct and indirect contact with humans. Diseases affecting possums are relatively poorly understood, as is the role of possums in the maintenance and transmission of zoonotic pathogens. This study aimed to investigate the significance of these possums as host species for a select range of pathogens, particularly in Victoria. Opportunistically collected serum samples from common ringtail, common brushtail and mountain brushtail possums (Trichosurus cunninghami) from Victoria and South Australia were tested for antibodies against Ross River virus (RRV) and flaviviruses. Cloacal swabs from common brushtail and ringtail possums from Victoria and New South Wales were screened for the presence of Mycobacterium ulcerans (the cause of Buruli ulcer), Campylobacter spp. and Salmonella spp. Spleen samples were screened for Francisella tularensis (the cause of tularaemia) and Coxiella burnetii (the cause of Q fever) by PCR. Antibodies to RRV were detected in 6.2% (16/259) of all possum samples, which were sampled during a concurrent epidemic of RRV in humans. Geographical areas with a moderate to high human case rate were associated with a higher seroprevalence rate in sampled possums. There was a significantly lower rate of detection in possums from urban environments compared to rural and mountainous environments. Seroconversion to an unspecified flavivirus was also present in 5.2% (5/97) of common ringtail possums and 6.5% (2/31) of mountain brushtail possums, but not in common brushtail possums (0/137). Two of the seropositive animals displayed