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One hundred <i>Prunus</i> trees, including almond (<i>P. dulcis</i>), apricot (<i>P. armeniaca</i>), nectarine (<i>P. persica var. nucipersica</i>), peach (<i>P. persica</i>), plum (<i>P. domestica</i>), purple leaf plum (<i>P. cerasifera</i>) and sweet cherry (<i>P. avium</i>), were selected from growing regions Australia-wide and tested for the presence of 34 viruses and three viroids using species-specific reverse transcription-polymerase chain reaction (RT-PCR) or polymerase chain reaction (PCR) tests. In addition, the samples were tested using some virus family or genus-based RT-PCR tests. The following viruses were detected: <i>Apple chlorotic leaf spot virus</i> (ACLSV) (13/100), <i>Apple mosaic virus</i> (ApMV) (1/100), <i>Cherry green ring mottle virus</i> (CGRMV) (4/100), <i>Cherry necrotic rusty mottle virus</i> (CNRMV) (2/100), <i>Cherry virus A</i> (CVA) (14/100), <i>Little cherry virus 2</i> (LChV2) (3/100), <i>Plum bark necrosis stem pitting associated virus</i> (PBNSPaV) (4/100), <i>Prune dwarf virus</i> (PDV) (3/100), <i>Prunus necrotic ringspot virus</i> (PNRSV) (52/100), <i>Hop stunt viroid</i> (HSVd) (9/100) and <i>Peach latent mosaic viroid</i> (PLMVd) (6/100). The results showed that PNRSV is widespread in <i>Prunus</i> trees in Australia. Metagenomic high-throughput sequencing (HTS) and bioinformatics analysis were used to characterise the genomes of some viruses that were detected by RT-PCR tests and <i>Apricot latent virus</i> (ApLV), <i>Apricot vein clearing associated virus</i> (AVCaV), <i>Asian Prunus Virus 2</i> (APV2) and <i>Nectarine stem pitting-associated virus</i> (NSPaV) were also detected. This is the first report of ApLV, APV2, CGRMV, CNRNV, LChV1, LChV2, NSPaV and PBNSPaV occurring in Australia. It is also the first report of ASGV infecting <i>Prunus</i> species in Australia, although it is known to infect other plant species including pome fruit and citrus.