Reconstructing Australia’s pre-European mammal communities is challenging as many species became extinct, or were severely impacted on, prior to intensive scientific interest. Critical baseline data as a result, is almost non-existent, and alternative sources of information must be used, such as from Holocene fossil deposits. Cave-roosting owls are well known contributors of small mammal remains to Holocene deposits. To determine the original small mammals of the north western Flinders Ranges in South Australia, we examined well-preserved subfossil barn owl pellets and disarticulated pellet remains from the rock pile and sediment floor of a small cave in Aroona Dam near Leigh Creek. The sheltered depositional environment of the cave provided a unique sample of intact pellets that acted as a time capsule for the original small mammals of the area and effectively sealed off from any modern additions. The pellets were radiocarbon dated to between 678 and 1547 yrs BP. A total of 22 mammal species were identified from the assemblage. Over 34% of these species are now completely extinct, with over 21% locally extinct from the surrounding area. When data from our study is combined with existing Holocene records for the north western Flinders Ranges, it is evident there has been an overall drop in small mammal species diversity of around 56% since European settlement. Subfossil deposits in caves are useful indicators of the original mammal assemblages in Australia and provide critical data that are relevant to conservation of vulnerable species.