Bats (order Chiroptera) represent more than 20 % of all mammalian species on Earth and are reservoir hosts for a range of viruses that are deadly in humans and livestock. Understanding how bats tolerate dangerous viruses is a matter of intense research. We have identified microRNAs (miRNAs) from the transcriptome of the Black Flying Fox (Pteropus alecto), a host of Hendra virus. Small RNAs from four adult Pteropus alecto males were isolated and sequenced using Illumina RNA deep sequencing. The miRDeep2 miRNA prediction tool identified 453 miRNAs in the transcriptome data set, of which 156 were classified as “novel” – not identical to miRNAs existing in the miRBase database. A number of novel miRNAs were present and responsive to Hendra virus infection in bat cells in vitro, suggesting a role in bat antiviral or innate immune responses. A range of target genes for novel microRNAs were predicted. This study is the first to identify microRNAs relevant to the bat antiviral response, a process that may underpin the ability of bats to harbour deadly viruses.