Denning sites provide an important opportunity for the direct and indirect transmission of parasites between host animals. Nonetheless, we know little about the species and numbers of external parasites (ectoparasites) found in the dens of Australian marsupials. This study monitored ectoparasite prevalence and abundance in nest boxes mounted for the use of Trichosurus spp. at Boho South, south-eastern Australia over two years. The content of nest boxes was checked for ectoparasites every 5 weeks for the first 12 months, and then every 3 months for the subsequent 12 months, and the identity of mammal species having used the nest box was determined using hair samples. A wide range of ectoparasitic mites, including Trichosurolaelaps spp., Haemolaelaps spp. and Ornithonyssus spp., and ticks, including Ixodes tasmani and I. trichosuri was detected. The prevalence and abundance of mite species differed from that usually found when recovering them directly from Trichosurus spp. This study highlights the importance of including the host animal’s environment in assessments of ectoparasite exposure.