The diet of the Eastern Barn Owl Tyto javanica was investigated by examination of two samples of pellets (n = 11 and 39), pellet debris and prey remains from: (1) an occupied nest with fledgling in May 2009, and (2) an Owl’s winter roost in August 2009 near Tamworth in the grain belt of New South Wales. The breeding diet consisted, by number, of 91% mammals (90% rodents, including 87% House Mice Mus musculus) and 9% common farmland birds (n = 116 food items). The winter diet consisted of 99% House Mice and 1% bird (n = 188 food items). The fledgling Owl was killed when it failed to flush from its nest hollow as the tree was being felled, during approved clearing of the now Critically Endangered White Box–Yellow Box–Blakely’s Red Gum Grassy Woodland and Derived Native Grassland. Given the dependence of the Barn Owl on House Mice, and the ongoing clearing of hollow trees, we note the potential for (a) secondary poisoning of owls by rodenticides, especially during plagues of the invasive House Mouse in the grain belt, and (b) the Barn Owl’s long-term decline in abundance in New South Wales.