Flying-foxes (genus Pteropus) are the natural host of Hendra virus (HeV) which sporadically causes fatal disease in horses and humans in Australia. While there is strong evidence that urine is an important infectious medium that likely drives bat to bat transmission, and in all likelihood bat to horse transmission, we are less certain about the relative importance of alternative routes of excretion. Differentiating between alternative modes of transmission is critical in determining transmission rates in host-pathogen models. Determining the main routes of HeV excretion in flying-foxes is also important when assessing the relative risk of spillover to horses at the bat-horse interface. The main aim of this study was to determine the primary routes of HeV excretion in three of the four Australian flying-fox species, namely P. alecto, P. poliocephalus and P. scapulatus. A total of 2840 flying-foxes were captured and sampled between 2012 and 2014. A range of biological samples (urine; serum; urogenital, nasal, oral and rectal swabs) were tested for HeV using RT-qPCR. Forty-two P. alecto had HeV genome detected in at least one sample and were classified as “HeV-positive”. The 42 HeV-positive P. alecto returned a total of 78 positive samples, at an overall detection rate of 1.76% across all samples tested in this species (78/4436). Urine was the most sensitive sample for detecting HeV genome, with fewer detections in serum, nasal, oral and rectal swabs. There were no detections in P. poliocephalus (n = 1168 animals; n = 2699 samples) or P. scapulatus (n = 262 animals; n = 985 samples). While it has been widely assumed that all four species play an important role in HeV infection dynamics, recent evidence suggests that Black and Spectacled flying-foxes may be the main reservoir hosts; the results of this study are consistent with this hypothesis.